Milk-derived bioactive peptides protect against oxidative stress in a Caco-2 cell model.

Milk and milk-derived products are a relevant source of bioactive peptides, which are also potential components of functional foods. Bioactive peptides exert multiple actions including an antioxidant role. In the present paper, four synthetic peptides (NPYVPR, AVPYPQR, KVLPVPEK, and ARHPHPHLSFM), corresponding to milk-derived peptides were studied. Although with different features, as revealed by RP-HPLC chromatography and MS analysis, the obtained peptides were shown to be taken up by Caco-2 cells arranged in an epithelial monolayer formation. The four peptides were all able to preserve cell viability against induced oxidative stress indicating that they might have a role in the control of oxidative stress. Therefore, an estimation of total thiols and glutathione content was performed after cell treatment with oxidants like hydrogen peroxide (H2O2) or tert-butyl hydroperoxide (TbOOH). The peptides were able to prevent the decrease of both total thiols and glutathione induced by H2O2 or TbOOH, and, in addition, they showed a protective effect on the thiol-related antioxidant enzymes thioredoxin reductase and glutathione reductase. Finally, they caused a decrease of ROS production induced by TbOOH in Caco-2 cells. The reported results highlight the relevant antioxidant role played by bioactive peptides in cells, which adds to other previously known properties.

Effect of metal complexes on thioredoxin reductase and the regulation of mitochondrial permeability conditions.

Gold(I) compounds such as auranofin, chloro(triethylphosphine) gold(I), and aurothiomalate act on mitochondrial functional parameters by determining an extensive permeability transition and a decrease of membrane potential. On the contrary, pyridine nucleotides and glutathione are not modified, whereas a slight but significant decrease of total thiols is apparent. The effect of gold(I) compounds is essentially referable to the inhibition, in the nanomolar range, of thioredoxin reductase activity and to an increase of hydrogen peroxide production. Metal ions and metal complexes (zinc and cadmium acetate, cisplatin, tributyltin) are also good inhibitors of thioredoxin reductase, although in the micromolar range, and in addition, they act as inducers of permeability transition and of membrane potential decrease. At variance with gold(I) compounds, which appear to work almost exclusively on thioredoxin reductase, metal ions and complexes are less specific, since they are active on different mitochondrial targets, including the respiratory chain.

Combined effect of propofol and GSNO on oxidative phosphorylation of isolated rat liver mitochondria.

Isolated rat liver mitochondria have been treated with the general anaesthetic propofol (2,6-diisopropylphenol, 200 microM) and the physiological NO donor nitrosoglutathione (GSNO, 200 or 250 microM). The efficiency of the oxidative phosphorylation has been evaluated by measuring the respiration and ATP synthesis rates and the behavior of transmembrane electrical potential. In mitochondria energized by succinate, the simultaneous presence of both propofol and GSNO gives rise to a synergic action in affecting the resting and the ADP-stimulated respiration, the respiratory control ratio, the ATP synthesis, and the formation and utilization of the electrochemical transmembrane potential.

Oxidation of adrenaline and its derivatives by S-nitrosoglutathione.

An oxidizing effect of S-nitrosoglutathione toward adrenaline and its cyclic derivatives (adrenochrome and adrenolutin) is reported. The oxidation was monitored either spectrophotometrically or as oxygen uptake. Adrenaline was first oxidized to adrenochrome that, after isomerization to adrenolutin, was further oxidized to products monitored as fluorescence decrease. To occur to a significant extent, this oxidation requires copper ions that, in addition to a direct effect on the oxidation of the ortho-diphenol moiety, are also able to decompose nitrosothiols, giving rise to nitric oxide. The latter, after interaction with oxygen and superoxide, produces nitrogen oxides and peroxynitrite, respectively, that are important contributors to the oxidative process. In this context, catecholamines might act as regulatory factors toward nitric oxide and its derivatives.

The interactions of trialkyltin compounds with lysosomes from rat liver.

Interactions of two trialkyltin compounds with the lysosomes from a rat liver have been studied. It is shown that these compounds induce a fast alkalinisation in the matrix of energised lysosomes. The fast alkalinisation rate is similar to the one obtained with uncouplers of the oxidative phosphorylation. An identical effect has been obtained with lysosomes energised in a chloride-free medium. This supports the hypothesis that trialkyltin compounds behave not only as Cl-/OH- exchangers, but also as proton carriers in biological membranes. This result could explain the toxicity and in particular the neurotoxicity of trialkyltin compounds.

Virtual instrumentation for pH measurements in biological systems.

In the present communication a personal computer control methodology for pH data acquisition and analysis in biological systems is reported. The instrumental control, acquisition, storage, processing and presentation of the experimental data are provided by a data acquisition board, a graphical programming software and numerical analysis/graphics software. The major objective of this work is to improve the performance and flexibility of the personal computer acquisition system compared with traditional approaches depending on potentiometric recorders. In particular, virtual instruments for interfacing pH meters of different brands to a personal computer and for measuring proton changes in lightly buffered solutions during enzymatic reactions are provided.

Mitochondrial permeability transition and release of cytochrome c induced by retinoic acids.

Retinoic acids, structurally related to vitamin A, inhibit the in vitro proliferation of different types of normal and neoplastic cells. The effects of all-trans, 9-cis, and 13-cis retinoic acids were tested on mitochondria isolated from rat liver. All the compounds were able to induce the membrane permeability transition observed as swelling and decrease in membrane potential, but 13-cis retinoic acid appeared to be the most effective. The latter was also shown to stimulate the release of cytochrome c from mitochondria, suggesting a potential target of retinoids in the induction of cell apoptosis. Interestingly, EGTA and cyclosporin A, which strongly inhibit the permeability transition induced by 13-cis retinoic acid, were without effect on the release of cytochrome c from the mitochondrial intermembrane space.

Effect of 2,6-diisopropylphenol and halogenated anesthetics on tetraphenylphosphonium uptake by rat brain synaptosomes: determination of membrane potential.

The effect of 2,6-diisopropylphenol (propofol) in comparison to that of the halogenated anesthetics enflurane, isoflurane, and halothane on tetrapenylphosphonium uptake by rat brain synaptosomes was studied. A direct method to separately measure the synaptosomal and the mitochondrial transmembrane potential by using the tetraphenylphosphonium cation (TPP+) was utilized. The latter is a lipophylic charged molecule which distributes between two compartments according to the transmembrane electrical potential in the presence or absence of 60 mM KCl as a synaptosomal membrane depolarizing agent. After previously reporting the damages induced by general anesthetics on isolated mitochondria, the aim of this paper was to study their possible action on the synaptosomal membrane potential and whether or not drugs concentrations damaging isolated mitochondria are also effective on synaptosomal mitochondria. The results indicated that, in the presence of glucose, mitochondria included in synaptosomes were able to maintain a transmembrane potential of 202+/-8 mV (mean +/- SD) while the synaptosomal membrane showed a potential of 78+/-8 mV (mean +/- SD). When anesthetic concentrations (0.6-1 mM propofol, 10-40 microM enflurane, 30-50 microM isoflurane, 8-15 microM halothane) that impair mitochondrial energy metabolism were used, the synaptosomal transmembrane potential was maintained and, in addition, a slight increase of the TPP+ taken up was observed as the anesthetic concentration was increased.

An in vitro study on the toxic effects of nonylphenols (NP) in mitochondria.

This paper is focused on alkylphenols, compounds which are formed by the biodegradation of polyethoxilatedalkylphenols detergents. Our experiments show that alkylphenols act not only as detergents, but also as uncouplers of the oxidative phosphorylation. This effect, can be observed at very low doses, thus suggesting that the preferential target of nonylphenols in living organisms are mitochondria.

The role of adrenochrome in stimulating the oxidation of catecholamines.

Adrenochrome, a stable oxidation product formed after oxidation of adrenaline, strongly stimulates oxygen uptake occurring during the autoxidation of adrenaline, other catecholamines and ascorbate. Oxygen consumed is converted to hydrogen peroxide suggesting the occurrence of a redox cycling process. The reduction of adrenochrome operated by adrenaline is accelerated by the exclusion of oxygen indicating that the oxidation of adrenaline occurs directly and superoxide anion does not necessarily mediate it. Oxygen consumption, observed in the catecholamine/adrenochrome and ascorbate/adrenochrome systems, is due to the autoxidation of leucoadrenochrome that, at variance with adrenaline, easily autoxidizes also at physiological pH. Therefore, in these systems, leucoadrenochrome appears to be the major determinant of the production of superoxide anion.

Antioxidant properties of clozapine and related neuroleptics.

The antioxidant properties of clozapine and other related molecules were evaluated with the crocin bleaching test both in aqueous and non-aqueous environment. The tests of microsomal lipid peroxidation and carbonyl formation were also used. In aqueous solution, chlorpromazine and trifluoperazine appear particularly effective in the bleaching of crocin, while serotonin has an efficacy intermediate between those of phenothiazines and clozapine. The latter drug, on the other hand, in a non-aqueous medium shows an antioxidant power comparable to that of butylated hydroxytoluene, indicating that its antioxidant properties are better expressed in a hydrophobic environment of the type present in a biological membrane. In fact, in lipid peroxidation induced in microsomal membranes, clozapine, chlorpromazine, trifluoperazine and serotonin act as very good antioxidants; at low concentrations, clozapine appears to be the most efficient after butylated hydroxytoluene. Similarly, all these compounds markedly inhibit protein carbonyl formation, clozapine being one of the most efficient. Thus, under different in vitro experimental conditions, the neuroleptic drugs chlorpromazine and trifluoperazine and the antipsychotic substance clozapine act as very effective antioxidants; this property might, at least in part, be responsible for the physiological and clinical effects observed in vivo.

Interactions of trialkyllead compounds with rat liver mitochondria.

The interactions of two trialkyllead (TAL) compounds, (trimethyl)Pb-Cl and (tributyl)Pb-Cl, with mitochodria from rat liver have been studied. A stimulation of the respiratory rate induced by the trialkyllead compounds added at low doses was observed which was not dependent on the presence of chloride in the medium. In contrast with the major current view, we propose that trialkyllead compounds behave as uncouplers of the oxidative phosphorylation and not (or not only) as Cl-/OH- exchangers. In fact the present results suggest that the TAL compounds enter the mitochondria as (alkyl)3Pb+ cations and are extruded as electroneutral (alkyl)3 Pb-OH compounds, the overall result being the transport of a proton through the membrane as in the case of classical uncouplers. The uncoupling effect could explain the toxicity of the compounds as a result of the decrease in the energy level of the cell. Furthermore, such a mechanism, in which the uptake of TAL compounds is supposed to be driven by a negative potential, could explain their preferential toxicity for neuronal cells, which maintain a higher negative-inside potential than most other cell types.

Mitochondrial effects of L-ropivacaine, a new local anesthetic.

The effects of the local anesthetics ropivacaine and bupivacaine were investigated on isolated rat liver mitochondria. The efficiency of oxidative phosphorylation was evaluated by measuring the rates of respiration and ATP synthesis and the magnitude of the transmembrane electrical potential (deltapsi). Bupivacaine did not alter the ADP-stimulated respiration but strongly affected the resting respiration, which was more than doubled at 0.6 mM. In addition, it decreased the transmembrane electrical potential, and the ATP synthesis rate (deltapsi was less than 100 mV at 0.6 mM). Ropivacaine did not alter the ADP-stimulated respiration, and the resting respiration seemed to be substantially unaffected up to 1.2 mM; a slight increase was observed at 1.8 and 2.4 mM. The transmembrane potential was decreased by anesthetic concentrations higher than 1.2 mM and ATP synthesis was consequently affected. The findings suggest that ropivacaine is less toxic than bupivacaine, in rat liver mitochondria.

Effect of the intravenous anesthetic 2,6-diisopropylphenol on respiration and energy production by rat brain synaptosomes.

The sensitivity of the mitochondrial energy production system to propofol (DPP) has been investigated in rat brain synaptosomes. DPP at 0.8 mM concentration produced a partial inhibition of coupled respiration, an apparent decrease of the oxygen uptake stimulation induced by CCCP and a full inhibition of the mitochondrial ATP production by synaptosomes. Higher concentrations of DPP (1 mM) fully abolish uncoupler-dependent stimulation and at 1.3 mM DPP also coupled respiration is completely blocked. Similar results were obtained when dinitrophenol replaced CCCP and phenol or propylbenzene replaced DPP. The presence of the alkyl residues seems critical for the DPP effect. In the presence of 30 mM glutamate both respiration and ATP production are enhanced but DPP effects are similar to those obtained in the absence of glutamate.

Influence of the anesthetic 2,6-diisopropylphenol (propofol) on isolated rat heart mitochondria.

The influence of the anesthetic 2,6-diisopropylphenol on isolated rat heart mitochondria has been investigated at a range of concentrations encompassing high and low clinical values. Low clinical concentrations of the anesthetic appeared unable to affect both oxidative phosphorylation and calcium homeostasis. 2,6-diisopropylphenol at high clinical levels decreased both the transmembrane electrical potential and the synthesis of ATP, while leaving mitochondrial calcium homeostasis unaffected. The results obtained suggest that isolated heart mitochondria are substantially insensitive to low clinical concentrations of 2,6-diisopropylphenol, thus largely excluding the possibility that mitochondrial alterations might be involved in the cardiac depression induced by this anesthetic.

Alteration of mitochondrial bioenergetics due to intravenous injection of a perfluorocarbon emulsion.

Wistar albino rats were intravenously injected with 1 ml of an oxyphoretic emulsion of perfluorobutyl-furane and killed 3, 7 or 30 days later. Mitochondria isolated from the liver and kidneys of treated rats showed a small decrease in the transmembrane electrical potential and a substantial depression of the rates of both ATP synthesis and ADP-stimulated respiration. These alterations in mitochondrial oxidative phosphorylation appear to be induced by perfluorocarbon and/or tensioactive molecules interacting with hydrophobic cell structures.

Effects of diltiazem on the calcium accumulation and ATP synthesis simultaneously sustained by isolated rat heart mitochondria.

1. The effects of diltiazem have been investigated in isolated rat heart mitochondria exposed to conditions possibly attained in ischemia-damaged cells. 2. The results obtained indicate that diltiazem, at the concentrations expected within cells following pharmacological treatment, does not significantly affect the mitochondrial calcium content. 3. Diltiazem did not appear to modify ATP synthesis, and hence the capacity of mitochondria to sustain the ATP-requiring processes needed for the recovery of cardiac cells.

Effects of diltiazem on liver mitochondria of rats: a reconsideration.

1. The effects of the Ca-channel blocker diltiazem (a drug of the benzothiazepine family) on bioenergetic metabolism have been assessed on isolated rat liver mitochondria. 2. Millimolar concentrations of diltiazem induced a decrease of both the ADP- and the uncoupler-stimulated respiration and a concomitant slight increase of the resting respiration. 3. Under the same experimental conditions diltiazem decreased the transmembrane electrical potential while leaving calcium uptake unaffected. 4. Micromolar concentrations of diltiazem, which are close to therapeutic haematic levels, were without effect.