Antioxidant effect of diethyldithiocarbamate on microsomal lipid peroxidation assessed by low-level chemiluminescence and alkane production

Different thiol-containing compounds, such as diethyldithiocarbamate (DDC), glutathione, penicillamine, and dithioerythritol have been chosen to study their effect on ascorbate/Fe-ADP-induced lipid peroxidation, detected by low-level chemiluminescence and alkane production. In the concentration range used, these thiols exerted a temporary protection against lipid peroxidation by lengthening the induction period; after overcoming this induction period, no substantial inhibition of either chemiluminescence or alkane production was observed. DDC was effective in protecting against lipid peroxidation in the nanomolar range, whereas the group of other thiol-containing molecules operated in the millimolar range.     

Hydroperoxide-induced chemiluminescence in rabbit lungs: role of arachidonic acid enzymes

Low-level chemiluminescence (C) is thought to be an index of oxidant stress. We measured the relationship between low-level C, pulmonary arterial pressure, and perfusate concentration of thromboxane B2 (TxB2) in isolated perfused rabbit lungs during challenge with tert-butyl hydroperoxide (t-bu-OOH). We also measured glutathione release as another index of oxidant stress. We found that C was correlated with each variable, suggesting that oxidant stress measured by C and by glutathione release stimulated TxB2 production and pulmonary vasoconstriction. We also investigated the contribution of active O2 metabolites produced by prostaglandin (PG) peroxidase to oxidant stress by studying the effects of t-bu-OOH before and after the use of cyclooxygenase and lipoxygenase inhibitors. We found that C was augmented after inhibition, perhaps due to metabolism of t-bu-OOH by peroxidases of both arachidonic acid (AA) metabolic pathways in the absence of their normal substrates. We studied phenylbutazone, thought to inhibit peroxidases, and AA. C during t-bu-OOH administration was not augmented after phenylbutazone and was markedly inhibited after AA administration perhaps because AA competes with t-bu-OOH. To further study the role of peroxidases we pretreated the lungs with the antioxidant dithiothreitol, which inhibits peroxidases involved in both the cyclooxygenase and lipoxygenase pathways. Dithiothreitol nearly abolished C produced by t-bu-OOH and also prevented the increased light caused by eicosatetrynoic acid. We directly tested the hypothesis that C occurred as a result of the interaction of t-bu-OOH and the cyclooxygenase and lipoxygenase enzymes; we measured C when t-bu-OOH was added to purified PGH2 synthase or soybean lipoxygenase. The combination of t-bu-OOH with PGH2 synthase or lipoxygenase led to C that was inhibited by dithiothreitol and by the antioxidant phenol. These results suggest that enzymes involved in AA metabolism can interact with t-bu-OOH and that the action of these enzymes on t-bu-OOH leads to C. The results may mean that lipid peroxides can indirectly contribute to tissue oxidant stress due to production of active O2 metabolites as by-products of their metabolism by AA peroxidases.     

Antioxidant Activity of 5-Hydroxytryptophan, 5-Hydroxyindole, and Dopa Against Microsomal Lipid Peroxidation and Its Dependence on Vitamin E

The antioxidant capacity of 5-hydroxy-tryptophan. 5-hydroxy-indole. and DOPA (3,4-dihydroxy-phenyI-alanine) was tested in the Fe-induced lipid peroxidation of liver microsomes of normal- and vitamin E-deficient rats, using ascorbate as a reductant. Lipid peroxidation was monitored as low-level chemilu-minescence, indicative of generation of electronically-excited states arising from the recombination of secondary lipid peroxyl radicals.     

Glutathione-dependent reduction of peroxides during ferryl- and met-myoglobin interconversion: a potential protective mechanism in muscle

Met-myoglobin is oxidized both by H2O2 and other hydroperoxides to a species with a higher iron valency state and the spectral characteristics of ferryl-myoglobin. Glutathione (GSH) reduces the latter species back to met-myoglobin with parallel oxidation to its disulfide (GSSG) but cannot reduce met-myoglobin to ferrous myoglobin. Under aerobic conditions, the GSH-mediated reduction of ferry-myoglobin is associated with O2 consumption and amounts of GSSG are formed far in excess over that of the peroxide added. Under anaerobic conditions, this ratio is close to unity. These results are interpreted in terms of a one-electron redox process involving the reduction of ferryl-myoglobin to met-myoglobin and the one-electron oxidation of GSH to its thiyl radical. Further reactions of thiyl radicals are influenced by the presence of oxygen which will be the determining factor in the ratio H2O2 added/GSSG formed. It is suggested that, when oxygen is limiting, myoglobin may serve as a protector of muscle cells against peroxides and other oxidants.     

Increase in heart glutathione redox ratio and total antioxidant capacity and decrease in lipid peroxidation after vitamin e dietary supplementation in guinea pigs

Dietary treatment with three diets differing in vitamin E, Low E (15 mg of vitamin E/kg diet), Medium E (150 mg/kg), or High E (1,500 mg/kg), resulted in guinea pigs with low (but nondeficient), intermediate, or high heart a-tocopherol concentration. Neither the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and reductase, nor the nonenzymatic antioxidants, GSH, ascorbate, and uric acid were homeostatically depressed by increases in heart a-tocopherol. Protection from both enzymatic (NADPH dependent) and nonenzymatic (ascorbate-Fe²⁺) lipid peroxidation was strongly increased by vitamin E supplementation from Low to Medium E Whereas no additional gain was obtained from the Medium E to the High E group. The GSH/GSSG and GSH/total glutathione ratios increased as a function of the vitamin E dietary concentration closely resembling the shape of the dependence of heart a-tocopherol on dietary vitamin E. The results show the capacity of dietary vitamin E to increase the global antioxidant capacity of the heart and to improve the heart redox status in both the lipid and water-soluble compartments. This capacity occurred at levels six times higher than the minimum daily requirement of vitamin E, even in the presence of optimum dietary vitamin C concentrations and basal unstressed conditions. The need for vitamin E dietary supplementation seems specially important in this tissue due to the low constitutive levels of endogenous enzymatic and nonenzymatic antioxidants present of the mammalian heart in comparison with those of other internal organs.     

Age-Dependent Modulation of Synaptic Plasticity and Insulin Mimetic Effect of Lipoic Acid on a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease is a progressive neurodegenerative disease that entails impairments of memory, thinking and behavior and culminates into brain atrophy. Impaired glucose uptake (accumulating into energy deficits) and synaptic plasticity have been shown to be affected in the early stages of Alzheimer’s disease. This study examines the ability of lipoic acid to increase brain glucose uptake and lead to improvements in synaptic plasticity on a triple transgenic mouse model of Alzheimer’s disease (3xTg-AD) that shows progression of pathology as a function of age; two age groups: 6 months (young) and 12 months (old) were used in this study. 3xTg-AD mice fed 0.23% w/v lipoic acid in drinking water for 4 weeks showed an insulin mimetic effect that consisted of increased brain glucose uptake, activation of the insulin receptor substrate and of the PI3K/Akt signaling pathway. Lipoic acid supplementation led to important changes in synaptic function as shown by increased input/output (I/O) and long term potentiation (LTP) (measured by electrophysiology). Lipoic acid was more effective in stimulating an insulin-like effect and reversing the impaired synaptic plasticity in the old mice, wherein the impairment of insulin signaling and synaptic plasticity was more pronounced than those in young mice.     

Inactivation of a testis-specific Lis1 transcript in mice prevents spermatid differentiation and causes male infertility

Lis1 protein is the non-catalytic component of platelet-activating factor acetylhydrolase 1b (PAF-AH 1B) and associated with microtubular structures. Hemizygous mutations of the LIS1 gene cause type I lissencephaly, a brain abnormality with developmental defects of neuronal migration. Lis1 is also expressed in testis, but its function there has not been determined. We have generated a mouse mutant (LIS1GT/GT) by gene trap integration leading to selective disruption of a Lis1 splicing variant in testis. Homozygous mutant males are infertile with no other apparent phenotype. We demonstrate that Lis1 is predominantly expressed in spermatids, and spermiogenesis is blocked when Lis1 is absent. Mutant spermatids fail to form correct acrosomes and nuclei appear distorted in size and shape. The tissue architecture in mutant testis appears severely disturbed displaying collapsed seminiferous tubules, mislocated germ cells, and increased apoptosis. These results provide evidence for an essential and hitherto uncharacterized role of the Lis1 protein in spermatogenesis, particularly in the differentiation of spermatids into spermatozoa.     

Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O(2)*-) and hydrogen peroxide (H(2)O(2)). The electron transfer chain of mitochondria is a well documented source of H(2)O(2); however, the release of O(2)*- from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O(2)*-, elucidating the mechanisms underlying the release of O(2)*- from mitochondria into cytosol, and assessing the role of outer membrane voltage-dependent anion channels (VDACs) in this process. Isolated rat heart mitochondria supplemented with complex I or II substrates generate an EPR signal ascribed to O(2)*-. Inhibition of the signal in a concentration-dependent manner by both manganese-superoxide dismutase and cytochrome c proteins that cannot cross the mitochondrial membrane supports the extramitochondrial location of the spin adduct. Basal rates of O(2)*- release from mitochondria were estimated at approximately 0.04 nmol/min/mg protein, a value increased approximately 8-fold by the complex III inhibitor, antimycin A. These estimates, obtained by quantitative spin-trapping EPR, were confirmed by fluorescence techniques, mainly hydroethidine oxidation and horseradish peroxidase-based p-hydroxyphylacetate dimerization. Inhibitors of VDAC, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), and dextran sulfate (in a voltage-dependent manner) inhibited O(2)*- production from mitochondria by approximately 55%, thus suggesting that a large portion of O(2)*- exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O(2)*- in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments.     

Nitric oxide and cell signaling pathways in mitochondrial-dependent apoptosis

Nitric oxide, generated by endogenous nitric oxide synthases or nitric oxide donors, can promote or prevent apoptosis induced by diverse pro-apoptotic stimuli in cell culture models. Both mitochondrial-dependent and -independent apoptotic signaling pathways mediate this dichotomous cellular response to nitric oxide. The molecular mechanisms behind these effects are complex and involve a number of nitrogen oxide-related species that are more reactive than nitric oxide itself. The local cellular environment plays a dynamic role in determining the nature and concentration of these species. Important components of the microenvironment include: the cellular redox state, glutathione, transition metals and the presence of other oxygen- and nitrogen-centered radicals. In particular, redox-sensitive nitrosating species are favorably generated under physiological conditions and capable of modifying multiple cell signaling pathways through reversible S-nitrosation reactions. Cytochrome c release from mitochondria is an important mechanism for the activation of caspase-3 and the initiation of cell death in response to 'intrinsic' pro-apoptotic stimuli, including oxidative and nitrosative stress. In turn, caspases and mitogen associated protein kinases may modulate cytochrome c release through their effects on the Bcl-2 family of proteins. This review will focus on (i) the importance of the cellular environment in determining the fate of nitric oxide and (ii) the ability of S-nitrosation to regulate mitochondrial-dependent apoptosis at the level of mitochondrial bioenergetics, cytochrome c release, caspases, mitogen associated protein kinases, and the Bcl-2 family of proteins.     

Stability of an extreme halophilic alkaline phosphatase from Halobacterium salinarium in non-conventional medium

Alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) was solubilised in organic medium using reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The stability of alkaline p-nitrophenylphosphate phosphatase in this system was studied at different conditions, w(0) ([H(2)O]/[surfactant]), salt concentration, with and without Mn(+2). At all the conditions assayed, alkaline p-nitrophenylphosphate phosphatase was more stable in reversed micelles than in bulk aqueous solution (at 25 degrees C). The stabilisation effect of the reversed micelles was dramatic when the enzyme was dialysed against Mn(+2)-free buffer since the enzyme lost all the activity within 90 min in aqueous medium, but it retained approximately 72% of the initial enzymatic activity for 90 min in reversed micelles.