Concerted action of the renin-angiotensin system, mitochondria, and antioxidant defenses in aging

Angiotensin-converting enzyme inhibitors (ACEi) and AT-1 receptor blockers (ARB) are two types of drugs that inhibit the renin-angiotensin system (RAS), and can attenuate the progression to cardiac and/or renal functional impairment, secondary to diverse pathologies. Some of the beneficial effects of ACEi and ARB occur independently of the ability of these drugs to reduce arterial blood pressure. Both, in animals, and in humans, we observed an enhancement of antioxidant defenses that occurred after treatment with ACEi. Based on these results, we postulate that some of the beneficial health effects associated to RAS inhibition can be ascribed to the prevention of oxidant-mediated damage. Furthermore, considering that: (i). RAS inhibition attenuates certain age-associated degenerative changes; (ii). aging was postulated to result from the accumulation of oxidant-mediated damage; and (iii). mitochondria are a major source of oxidants, we studied potential associations among RAS inhibition, mitochondrial function and production of oxidants and nitric oxide, and aging. The results obtained suggest, that RAS inhibitors, i.e. enalapril and losartan, can protect against the effects of aging by attenuating oxidant damage to mitochondria, and in consequence, they preserve mitochondrial function. The mechanism(s) explaining such attenuation of oxidant damage can relay on a reduction of the ANG-II-dependent generation of superoxide and/or an increased detoxification of reactive nitrogen and oxygen species by recomposition of antioxidant defense levels.     

Selenium-induced antioxidant protection recruits modulation of thioredoxin reductase during excitotoxic/pro-oxidant events in the rat striatum

Selenium (Se) is a crucial element exerting antioxidant and neuroprotective effects in different toxic models. It has been suggested that Se acts through selenoproteins, of which thioredoxin reductase (TrxR) is relevant for reduction of harmful hydroperoxides and maintenance of thioredoxin (Trx) redox activity. Of note, the Trx/TrxR system remains poorly studied in toxic models of degenerative disorders. Despite previous reports of our group have demonstrated a protective role of Se in the excitotoxic/pro-oxidant model induced by quinolinic acid (QUIN) in the rat striatum (Santamaría et al., 2003, 2005), the precise mechanism(s) by which Se is inducing protection remains unclear. In this work, we characterized the time course of protective events elicited by Se as pretreatment (Na(2)SO(3), 0.625 mg/kg/day, i.p., administered for 5 consecutive days) in the toxic pattern produced by a single infusion of QUIN (240 nmol/μl) in the rat striatum, to further explore whether TrxR is involved in the Se-induced protection and how is regulated. Se attenuated the QUIN-induced early reactive oxygen species formation, lipid peroxidation, oxidative damage to DNA, loss of mitochondrial reductive capacity and morphological alterations in the striatum. Our results also revealed a novel pattern in which QUIN transiently stimulated an early TrxR cellular localization/distribution (at 30 min and 2 h post-lesion, evidenced by immunohistochemistry), to further stimulate a delayed protein activation (at 24 h) in a manner likely representing a compensatory response to the oxidative damage in course. In turn, Se induced an early stimulation of TrxR activity and expression in a time course that "matches" with the reduction of the QUIN-induced oxidative damage, suggesting that the Trx/TrxR system contributes to the resistance of nerve tissue to QUIN toxicity.     

Blood metalloproteins of pro-oxidant and antioxidant action in psoriasis]

Molecular mechanism of pathogenesis of psoriasis related with intensity of formation of oxygen active forms high concentration, depends on balance of pro-oxidant and antioxidant systems activity and leads to quantitative changes of toxic agents of peroxidation processes in blood. Observed results show disorders in Cu, Zn-superoxide dismutase activity and contents of ceruloplasmin, transferrin and new revealed pro-oxidant metalloproteins-cytochromes b558(III), b558(IV), b5, O2(-)-generating lipoprotein suprol and indicate of disturbances in intensity of lipid peroxidation processes. They are considered as obligatory but not specific link in molecular mechanisms of different etiology of oxidative stress formation being as important argument in pathogenesis of various diseases as well as psoriasis.     

Oxidative stress and reduced antioxidant defenses in peripheral cells from familial Alzheimer's patients

We have measured the levels of typical end products of the processes of lipid peroxidation, protein oxidation, and total antioxidant capacity (TAC) in skin fibroblasts and lymphoblasts taken from patients with familial Alzheimer's disease (FAD), sporadic Alzheimer's disease (AD), and age-matched healthy controls. Compared to controls, the fibroblasts and lymphoblasts carrying amyloid precursor protein (APP) and presenilin-1 (PS-1) gene mutations showed a clear increase in lipoperoxidation products, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE). In contrast, the antioxidant defenses of cells from FAD patients were lower than those from normal subjects. Lipoperoxidation and antioxidant capacity in lymphoblasts from patients affected by sporadic AD were virtually indistinguishable from the basal values of normal controls. An oxidative attack on protein gave rise to greater protein carbonyl content in FAD patients than in age-matched controls. Furthermore, ADP ribosylation levels of poly(ADP-ribose) polymerase (PARP) nuclear substrates were significantly raised, whereas the PARP content did not differ significantly between fibroblasts carrying gene mutations and control cells. These results indicate that peripheral cells carrying APP and PS-1 gene mutations show altered levels of oxidative markers even though they are not directly involved in the neurodegenerative process of AD. These results support the hypothesis that oxidative damage to lipid, protein, and DNA is an important early event in the pathogenesis of AD.     

Concentration dependent antioxidant/pro-oxidant activity of curcumin studies from AAPH induced hemolysis of RBCs

The antioxidant properties of curcumin have been studied by evaluating its ability to protect RBCs from AAPH (2,2'-azobis (2-amidinopropane) hydrochloride) induced oxidative damage. RBCs are susceptible to oxidative damage, resulting in peroxidation of the membrane lipids, release of hemoglobin (hemolysis), release of intracellular K(+) ions and depletion of glutathione (GSH). In this paper, lipid peroxidation, hemolysis and K(+) ion loss in RBCs were assessed respectively by formation of thiobarbituric acid reactive substances (TBARS), absorbance of hemoglobin at 532nm and flame photometry. The treatment of RBCs with curcumin showed concentration dependant decrease in level of TBARS and hemolysis. The IC(50) values for inhibition of lipid peroxidation and hemolysis were estimated to be 23.2+/-2.5 and 43+/-5microM respectively. However in contrast to the above mentioned effects, curcumin in similar concentration range, did not prevent release of intracellular K(+) ions during the process of hemolysis, rather curcumin induced its release even in the absence of hemolysis. The ability of curcumin to prevent oxidation of intracellular GSH due to hemolysis showed mixed results. At low concentrations of curcumin (<10microM) it prevented GSH depletion and at higher concentrations, the GSH levels decreased gradually. Curcumin scavenges the peroxyl radical generated from AAPH. Based on these results, it is concluded that curcumin exhibits both antioxidant/pro-oxidant activity, in a concentration dependent manner.     

Modulation of hOGG1 DNA repair enzyme in human cultured cells in response to pro-oxidant and antioxidant challenge

The putative modulation of the base excision repair enzyme, human 8-oxoguanine glycosylase (hOGG1), important in the removal of the potentially mutagenic lesion 8-oxo-2'-deoxyguanosine (8-oxodG), was investigated in human cell culture models. The expression of specific mRNA and protein was measured following pro-oxidant and antioxidant treatments in one human lymphoblastoid and one keratinocyte line. The measurement of intracellular reactive oxygen species generation was monitored by a fluorogenic assay and potential genotoxic effects confirmed by the dose-dependent increase in formamidopyrimidine-DNA glycosylase (Fpg) sensitive sites by alkaline unwinding following sub-lethal doses of hydrogen peroxide. The generation of a potentially antioxidant environment was assessed by the intracellular increase and extracellular depletion in ascorbic acid, confirmed by capillary electrophoresis. Despite these pro-oxidant and antioxidant treatments no significant change in mRNA of hOGG1 was observed in either cell line. Western analysis revealed that relatively high, yet noncytotoxic, doses of hydrogen peroxide caused a consistent approximate 50% decrease in hOGG1 protein in lymphoblastoid cells. The lack of upregulation of hOGG1 suggests the gene is constitutively expressed, which is further supported by studies examining the sequence of its promoter region. However, hOGG1 protein turnover may be sensitive to intracellular redox changes.     

The use of prostacyclin in the separation from plasma and washing of human platelets

A new method for the separation from plasma and washing of human platelets is described. The use of prostacyclin (PGI₂) throughout the procedure prevents the activation of platelets. The method allows a 60–70% yield of platelets from PRP. The platelet sensitivity to ADP, collagen, adrenaline, arachidonic acid and thrombin is the same as in PRP. The platelet suspension is stable for long periods and the reactivity to aggregating agents remain unchanged for periods greater than 48 h when platelets are stored at 4°C.     

Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm

Glutathione peroxidase is one of the principal antioxidant defense enzymes in human spermatozoa, but it requires oxidized glutathione to be reduced by glutathione reductase using NADPH generated in the pentose phosphate pathway. We investigated whether flux through the pentose phosphate pathway would increase in response to oxidative stress and whether glutathione reductase was required to protect sperm from oxidative damage. Isotopic measurements of the pentose phosphate pathway and glycolytic flux, thiobarbituric acid assay of malondialdehyde for lipid peroxidation, and computer-assisted sperm analysis for sperm motility were assessed in a group of normal, healthy semen donors. Applying moderate oxidative stress to human spermatozoa by adding cumene hydroperoxide, H(2)O(2), or xanthine plus xanthine oxidase or by promoting lipid peroxidation with ascorbate increased flux through the pentose phosphate pathway without changing the glycolytic rate. However, adding higher concentrations of oxidants inhibited both the pentose phosphate pathway and glycolytic flux. At concentrations of 50 microg/ml or greater, the glutathione reductase-inhibitor 1,3-bis-(2-chloroethyl) 1-nitrosourea decreased flux through the pentose phosphate pathway and blocked the response to cumene hydroperoxide. It also increased lipid peroxidation and impaired the survival of motility in sperm incubated under 95% O(2). These data show that the pentose phosphate pathway in human spermatozoa can respond dynamically to oxidative stress and that inhibiting glutathione reductase impairs the ability of sperm to resist lipid peroxidation. We conclude that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system is functional and provides an effective antioxidant defense in normal human spermatozoa.     

Fidelity of protein synthesis does not decline during aging of cultured human fibroblasts

To ascertain whether the fidelity of protein synthesis declines during cellular aging in vitro, we have developed a cell-free protein synthesizing system from cultured human fibroblasts which actively incorporates phenylalanine into acid-insoluble material upon addition of poly (U). The accuracy of poly(U)-directed protein synthesis was determined by comparing the ratio of leucine to phenylalanine incorporation in extracts of early- and late-passage fibroblasts derived from normal persons and from subjects with two genetic disorders of premature aging, progeria, and Werner syndrome. The results show no decline in translational fidelity at late passage or in prematurely aging cells, and thus fail to support the error catastrophe theory of cellular aging.     

Temporal separation of protein toxin translocation from processing events

Intoxication of Vero cells by ricin, modeccin, diphtheria toxin (DT), and Pseudomonas exotoxin A requires: 1) binding to cell surface receptors; 2) transport to the cytoplasm; and 3) enzymatic inactivation of a component of the protein synthetic machinery. The kinetic profiles of all four toxins consist of a lag followed by the apparent first-order decrease in protein synthesis. Autoradiographic analysis of DT-intoxicated cell populations has demonstrated that two subpopulations of cells exist during the period of decreasing protein synthesis: one population synthesizing at control levels and the other synthesizing little or no protein (Hudson, T. H., and Neville, D. M., Jr. (1985) J. Biol. Chem. 260, 2675-2680). The present study correlates the autoradiographic data with the rates of protein synthesis decline in cells intoxicated with modeccin, ricin, Pseudomonas exotoxin A, as well DT. In all cases, the first time point which exhibits a decrease in protein synthetic activity also exhibits two subpopulations of cells, one synthesizing protein at control rates and the other synthesizing little or no protein. As the intoxication progresses, cells leave the control population by the rapid cessation of all protein synthesis. These experiments demonstrate that transport of all four toxins to the cytosol is the rate-limiting step during the pseudo first-order decline in protein synthesis. Furthermore, the final step in the transport process (translocation) must result in the release to the cytoplasm of a quantity of toxin sufficient to rapidly inactivate all protein synthesis in that cell. The probability of a translocation event occurring in any cell of the population is established during the lag and remains constant throughout the first-order decrease in protein synthesis. The requirement for acidification during the intoxication by DT, Pseudomonas exotoxin A, or modeccin is restricted to the lag period. Acidification is therefore necessary to establish the probability of translocation, but it is not directly involved in the actual translocation of these toxins. The pseudo first-order passage of DT intoxications through antitoxin and NH4Cl- or monensin-sensitive stages are shown to have the same cellular basis as the pseudo first-order decrease in protein synthesis. A kinetic model is presented which defines the DT intoxication process from one of its earliest events (endocytosis) to its penultimate event (translocation of toxin to the cytosol).(ABSTRACT TRUNCATED AT 400 WORDS)     

Anti- and pro-oxidant effects of quercetin in copper-induced low density lipoprotein oxidation. Quercetin as an effective antioxidant against pro-oxidant effects of urate.

We recently reported that, depending on its concentration, urate is either a pro- or an antioxidant in Cu(2+)-induced low-density lipoprotein (LDL) oxidation. We also previously demonstrated an antioxidant synergy between urate and some flavonoids in the Cu(2+)-induced oxidation of diluted serum. As a result, the effect of the flavonoid quercetin on the Cu(2+)-induced oxidation of isolated LDL has been studied either in the presence or absence of urate. We demonstrate that, like urate, quercetin alone, at low concentration, exhibits a pro-oxidant activity. The pro-oxidant behavior depends on the Cu(2+) concentration but it is not observed at high Cu(2+) concentration. When compared with urate, the switch between the pro- and the antioxidant activities occurs at much lower quercetin concentrations. As for urate, the pro-oxidant character of quercetin is related to its ability to reduce Cu(2+) with the formation of semioxidized quercetin and Cu(+) with an expected yield larger than that obtained with urate owing to a more favorable redox potential. It is also shown that the pro-oxidant activity of urate can be inhibited by quercetin. An electron transfer between quercetin and semioxidized urate leading to the repair of urate could account for this observation as suggested by recently published pulse radiolysis data. It is anticipated that the interactions between quercetin-Cu(2+)-LDL and urate, which are tightly controlled by their respective concentration, determine the balance between the pro- and antioxidant behaviors. Moreover, as already observed with other antioxidants, it is demonstrated that quercetin alone behaves as a pro-oxidant towards preoxidized LDL.     

Isolation of antithrombin III from human plasma: its separation from alpha1-antitrypsin1.

Plasma was adsorbed with Al(OH)₃ in a ratio of 8 : 2. The gel was washed free of entrapped plasma and antithrombin III and α₁-antitrypsin eluted by repeated washing with 0.36 M ammonium phosphate, pH 8.1. The crude inhibitor preparation was subjected to chromatography on QAE-Sephadex A-50 at pH 8.0, followed by gel filtration on Sephadex G-200. In these two preparative steps the two inhibitors eluted together. However, they were separated by rechromatography on QAE-Sephadex at pH 7.4, following which they were recovered in highly purified form, α₁-antitrypsin by passage through concanavalin-A-Sepharose and antithrombin III through heparin-Sepharose.     

Affinity separation of human plasma gelsolin on Affi-Gel Blue

Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%.