Mechanisms involved in Pb2+-mediated enhancement of lipid oxidation
Viviana N. Adonaylo and Patricia I. Oteiza
Departamento de Química Biológica, IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina
Experimental evidences suggest that cellular damage mediated by oxidants could be involved in the pathology associated with lead (Pb) intoxication. We have investigated the effect of Pb2+ on lipid oxidation in liposomes using different initiators. In the presence of 100 µM Fe2+, Pb2+ (12.5-200 µM) stimulated lipid oxidation in phosphatidyl serine- and cardiolipin-containing liposomes, measured as 2-thiobar bituric acid-reactive substances (TBARS) and conjugated dienes. Kinetic studies showed that Pb2+ acted by decreasing the lag of Fe2+ initiated TBARS production. The capacity of Pb2+ to stimulate Fe2+ supported lipid oxidation was positively correlated with its capacity to promote membrane lateral phase separation. The effect of Pb2+ on TBARS production was also studied in the presence of other initiators. Pb2+ did not stimulate TBARS formation when lipid oxidation was initiated by the azocompounds 2,2'-azobis (2,4-dimethylvaleronitrile) and 2,2'-azobis (2-amidinopropane) (AAPH), lipid and water soluble free radical generators respectively. However, Pb2+ significantly stimulated TBARS production and NADH oxidation in the presence of a photochemical source of O2. These effects were inhibited (50 and 100% respectively) by superoxide dismutase. The present results suggest that Pb2+ can stimulate lipid oxidation at multiple level by: a) interacting with Fe2+, b) forming with O2 a complex with higher oxidizing capacity than O2 per se and/or,c) causing changes in membrane physical properties which would favor lipid oxidation.
Supported by grants from the Universidad de Buenos Aires (FA116, FA055), CONICET and Fundación Antorchas
Determination of the chain-breaking rate constant of antioxidants in complex biological systems
Fernando Antunes1,2, Lawrence Ross Coates Barclay3 and Ruy E. Pinto1
1Grupo de Bioquímica e Biologia Teóricas, Instituto Bento da Rocha Cabral, P-1250 Lisboa, Portugal, 2Molecular Pharmacology ant Toxicology, University of Southern California, Los Angeles, California, USA, 3Mount Allison University, Department of Chemistry, Sackville, NB, Canada EOA 3CO
The classic method to determine the inhibition rate constant (kinh) for chain-breaking antioxidants is the inhibited oxygen uptake method. In this method, the oxygen consumption during lipid peroxi dation is followed in the presence of a chain-breaking antioxidant. However, this method is very susceptible to interference from the chain-transfer reactions of the radicals of the antioxidant. To avoid the chain-transfer reactions the rate constant may be determined making use of the dependence of the ratio trans,cis-hydroperoxides/ trans,trans-hydroperoxides (t,c/t,t) on the hydrogen atom-donating ability of the medium. As proposed originally1, this method is applied by measuring the t,c/t,t ratio as a function of the concentration of the antioxidant. In this form, this method was recently applied for the first time to measure the kinh for a natural antioxidant, a-tocopherol.2 The value obtained was one order of magnitude higher than the value previously obtained by the inhibited oxygen uptake method. However, the measurement of the t,c/t,t ratio for different antioxidant concentrations implies the experimental control of this variable, which can only be done, under well-controlled conditions, in chemical systems. So, the determination of kinh in a complex biological system, like for example LDL particles, is not possible by measuring the t,c/t,t ratio as originally proposed. In the present work, a time-integrated method that allows the estimation of the kinh by following the t,c/t,t ratio with time, and therefore avoids the need to control the antioxidant concentration, is derived theoretically. The experimental application of this method is illustrated for a lipid bilayer.
1. Porter, N.A.; Lehman, L.S.; Weber, B.A.; Simth, K.J. J. Am. Chem. Soc. 103:6447-6455; 1981.
2. Barclay, L.R.C.; Vinqvist, M.R.; Antunes, F.; Pinto, R.E. J. Am. Chem. Soc. 119:5764-5765; 1997
Acknowledgements: FA acknowledges grants BD/5575/95 and BPD/11778/97 from PRAXIS XXI/JNICT.
K. Anzai, Y. Senzaki*, J. Ueda, Y. Miura, T. Ozawa, and Y. Matsushima*
National Institute of Radiological Sciences, Chiba, Japan and *Kyoritsu College of Pharmacy, Tokyo, Japan
Reactive oxygen species (ROS) are considered to cause various diseases through reactions with constituents of living body. It has been suggested that the formation of peroxidized lipids and their decomposed products play an important role in the harmful effect of ROS, such as induction of atherosclerosis and increase in membrane permeability. In the present study, we have investigated the effects of phospholipid composition on lipid peroxidation of liposomes made of asolectin or mixture of egg phosphatidylcholine (PC) and phosphatidylethanolamine (PE).
Lipid peroxidation reaction was initiated by the peroxyl radical (ROO .) or hydroxyl radical (.OH). ROO. was generated by the decomposition of AAPH and .OH was generated by X-irradiation or by the reaction of hydrogen peroxide (H2O2) with Cu2+ complex. The progress of the peroxidation reaction was estimated by the measurement of the absorbance at 234 nm, corresponding to the amount of conjugated diene, or the measurement of TBARS.
Constant application of the radicals increased the lipid peroxide but longer application induced the degradation of the lipid peroxide. Asolectin liposomes were more sensitive to the peroxidation than egg PC/PE liposomes because of larger amount of unsaturated fatty acyl chain in asolectin. When €OH was generated by the reaction of H2O2 with Cu2+ complex, increase of PE content in the liposomes accelerated the peroxidation reaction, whereas the increase of PE content had no effect on the peroxidation by €OH generated by X-irradiation. Absorption of Cu2+ on the surface of the liposomes through amino group of PE may be responsible for the acceleration
M.C. Arkan*, S. Camandola§, G. Leonarduzzi, G. Poli§, and H. Basaga*
*Bogazici University, Department of Molecular Biology and Genetics, Istanbul, Turkey, and §Torino University, Department of General Pathology, Turin, Italy
Oxidant and antixidant status of the cell can control gene activation through activator proteins such as NF-kB and AP-1. Direct redox effects on NF-kB and AP-1 have been reported for various cell lines. In our studies we have evaluated the effect of Asc/Fe3+ at different concentrations and time intervals, and detected the DNA binding activities of NF-kB and AP-1 by EMSA with either nuclear or cytoplasmic extracts obtained from J774A-1 cell line. Our results indicated that Asc/Fe3+ at high (1 mM 500 mM) and low (200 10 µM) concentrations led to decrease in the nuclear translocation of NF-kB and DNA-binding activity of AP-1. The oxidant DPPD when used as low as 5mM did not exert any effect in the DNA-binding activities of NF-kB and AP-1. PMA cotreatment with Asc/Fe3+ also led to a decrease in NF-kB nuclear translocation, in contrast an increased binding of AP-1 was observed. Nuclear binding of both transcription factors was increased when cells were treated with 1 mM HNE, the product of lipid peroxidation after 45 min. In order to better understand the mechanism of oxidative stress and its relationship with the activation of transcription factors we have determined GSH levels in Asc/Fe3+ treated cells. Significant levels of GSH were found to be depleted in experiments where cells were treated with 1mM-500mM Asc/Fe3+. Our results in agreement with the previous workers suggest that the intracellular GSH/GSSG levels may be important and GSH may function as a switch in the differential regulation of genes in response to oxidative signals.
Plant extracts as antioxidant prophylactic agents
Okezie I Aruoma
OICA International, P O Box MI 34, Micoud, Saint Lucia; and Pharmacology Group, King¹s College London, Manresa Road, London, SW3 6LX, UK
The shelf-life of manufactured foods is invariably limited by oxidative degradation of the polyunsaturated fatty acids of the food lipids and by browning reactions. That a role for free radicals and antioxidants in the process of aging and the pathogenesis of human diseases exists has led to the suggestion that antioxidants in particular, plant diet-derived antioxidants (including plant extracts with antioxidant indications) and drug-derived antioxidants, might have health benefits as prophylactic agents.
The need for food manufacturers to stabilize processedfoods by use of food-grade antioxidants during food processing is currently reflected by the increasing interest in the use of antioxidants as prophylactic agents in human degenerative diseases. Carnosol and carnosic acid accounts for over 90% of the antioxidant properties of rosemary extract. Both compounds are powerful inhibitors of lipid peroxidation either to membrane lipids or low density lipoproteins and are potent scavengers of peroxyl radicals and hypochlorous acid. Herbor 025, a rosemary extract containing carnosol and carnosic also stabilizes the food matrix against oxidation and could contribute to antioxidant protection in vivo.
Delineating the in vivo contribution of plant extracts and/or plant-derived antioxidants (the pure active principles in plant extracts with antioxidant indications), to the modulation of the pathological consequences of oxidative stress in the human body is a task whose time has come.
Annika Assmann, Karlis Briviba, and Helmut Sies
Heinrich-Heine-Universität Düsseldorf, Institut für Physiologische Chemie I, Postfach 101007, D-40001 Düsseldorf, Germany
Selenomethionine, an aminoacid occurring in proteins in place of methionine, reacts efficiently with oxidants, e.g. peroxynitrite, forming methionine selenoxide. The selenoxide is rapidly and efficiently reduced back to selenomethionine at the expense of two equivalents of glutathione in a nonenzymatic reaction1. In contrast, reduction of methionine sulfoxide requires an enzyme, methionine sulfoxide reductase.
Selenomethionine, but not methionine selenoxide, effectively protects against peroxynitrite-mediated oxidation of dihydrorhoda mine 123. However, in the presence of glutathione methionine selen oxide shows a protective effect similar to that of selenomethionine1. Similarly, reduction of methionine selenoxide to selenomethionine occurs in the presence of ascorbate, but in a much slower reaction. Ascorbate reduces methionine selenoxide in a 1:1 ratio, suggesting two electron reduction.
Thus, there is a low-molecular-weight defense system against peroxynitrite maintained by selenosubstituted methionine using biological reductants, e.g. glutathione or ascorbate.
1 Assmann, A., Briviba, K., and Sies, H. (1997). Arch. Biochem. Biophys., in press
J.E. Brown1, H. Khodr2, R.C. Hider2, and C.A. Rice-Evans1
1International Antioxidant Research Center UMDS-Guy¹s Hospital, London, SE1 9RT and 2Department of Pharmacy, King¹s College, University of London, London, SW3 6LX, United Kingdom
There is growing interest that flavonoids may be important dietary antioxidants. These compounds are found in almost every plant as well as in plant-derived beverages, such as tea and wine. Flavonoids act as antioxidants by scavenging reactive oxygen species, such as hydroxyl radical and superoxide anion, reactive nitrogen species, such as peroxynitrite and by intercepting lipid peroxyl radicals. Another mechanism by which flavonoids can display antioxidant activity is by chelating transition metal ionds. This aspect of flavonoid antioxidant behaviour in Cu(II)-mediated low-density lipoprotein (LDL) oxidation required further study. Therefore, the interaction of four structurally related flavonoids (quercetin, Kaempferol, rutin, and luteolin) with Cu(II) ions was investigated. This was examined in terms of the extent to which they undergo complex formation through chelation or modification through oxidation, and their structural dependence. It is evident that the o-3¹,4¹-di-OH substitution in the B ring is important for Cu(II)-chelate formation, thereby influencing the antioxidant activity. The presence of a 3-OH group in the flavonoid structure enhances oxidation of quercetin and kaempferol, whereas luteolin and rutin, each lacking the 3-OH group, do not oxidise as readily in the presence of Cu(II) ions.
| Flavonoid (OH) position | Cu(II) ion interaction | Lag phase | Kpart |
| Quercetin (3,5,7,3'4') | ++/oxidation | 387 +/- 35 | 1.20 +/- 0.1 |
| Rutin (5,7,3'4') | ++ | 284 +/- 13 | 0.37 +/- 0.1 |
| Luteolin (5,7,3'4') | ++ | 301 +/- 21 | 22.2 +/- 2.5 |
| Kaempferol (3,5,7,4') | +/oxidation | 170 +/- 9 | 69.5 +/- 1.3 |
Results expressed as Mean ± SD. Control lag phase = 100%.
The results demonstrated that the reactivities of the flavonoids in protecting LDL against Cu(II)-induced oxidation are dependent on their structural properties in terms of the response of the particular flavonoid to Cu(II) ions, whether chelation or oxidation, their partitioning abilities between aqueous and lipophilic environments, and their hydrogen-donating antioxidant properties.
This work was supported by The Ministry of Agriculture, Fisheries and Food.
Effect of ACEi on antioxidant defenses
Elena M.V. de Cavanagh1, Fernando Carrasquedo1, Daniel Scrivo2, A. Wassermann2, Leon Ferder3, Felipe Inserra3, Cesar G. Fraga1
1Physical Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires; 2CENHA; 3Institute of Biomedical Research-UHABI. Buenos Aires, Argentina
Angiotensin-converting enzyme inhibitors (ACEi) are widely used as therapeutic agents mainly in the treatment of hypertension, heart failure, coronary disease, and diabetic nephropathy. All these disease states have been related to oxidative conditions. We have found that enalapril and captopril can modulate antioxidant defenses in mice in vivo (FEBS Lett 1995;361:22-24, Am J Physiol 1997;272:R514 R518). Such modulation of antioxidant defenses (superoxide dismu tase, glutathione peroxidase, glutathione, total antioxidant capacity) was treatment-, and organ-dependent. Based on these results, we designed a clinical study in which it was determined that chronic renal patients in hemodialysis (CRP, n = 32) had lower levels of red blood cells GSH (RBC-GSH) and Se-dependent glutathione peroxidase activity (RBC-GPx) (25 and 60% of the values for healthy control subjects, respectively). Other antioxidant defenses were also diminished: a-tocopherol (14.8%); superoxide dismutase (12.6%); ubiqui nol-10 (42.5%). The cohort of these patients that was under treatment with enalapril (10 mg/day for at least six month; n =11) had RBC GSH and GPx that were 74 and 85% of the control subjects values, respectively. Plasma concentration of beta-carotene was lower in CRP as compared to controls (0.19 vs 0.35 µM) but it was significantly higher in the group treated with enalapril (0.54 µM). These results suggest that ACEi may protect tissues from oxidative damage by increasing enzymatic and nonenzymatic antioxidant defenses. The effect of ACEi on glutathione homeostasis underlies the maintenance of cells redox states as a potential mechanism for ACEi-antioxidant protection.
HIV-1 TAT decreases intracellular glutathione content in human Jurkat T cells
Jinah Choi and Henry Jay Forman
Department of Molecular Pharmacology & Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California
Human Immunodeficiency Virus (HIV-1) decreases glutathione (GSH) content in human. Furthermore, Tat-1, a transactivator encoded by HIV-1, is suggested to decrease GSH concentration in vitro, in Jurkat T cells as well as HeLa cells. Our present aim, therefore, was to determine whether HIV-1 Tat indeed caused a reduction in the GSH content in human T cells and the mechanism thereof. To this end, recombinant Tat protein (sTat) was added to human Jurkat clone E6.1 at ~1µg/ml concentration, and intracellular GSH content was monitored by HPLC. The control cells displayed a transient elevation in intracellular GSH content at 6 h after fresh seeding. Then, the GSH level gradually declined, approaching the initial level by 48 h. In sTat-treated cells, this elevation was suppressed, such that the GSH content was only 60 to 80 % of the control level at 6 h, without any corresponding rise in GSSG content. The difference in GSH content with sTat-treatment disappeared by 48 h. This effect of sTat could be prevented by preabsorption of sTat preparation with polyclonal antibodies and was reproduced with the commercially purchased sTat. Observed effect of sTat could not be attributed to the inhibition of g glutamyl transpeptidase activity or the cysteine unavailability. sTat, when added along with L-buthionine S,R-sulfoximine (BSO), did not lower GSH faster than BSO alone. There was no increase in apoptosis or necrosis with sTat. Moreover, 72 amino acid-sTat, lacking the second exon, resulted in a similar suppression of GSH elevation. Therefore, HIV-1 Tat decreased intracellular GSH level in human T cell line, independent of the second exon, most likely through a non oxidative mechanism that involves an inhibition of GSH biosynthesis.
Funded by the Dolores Zohrab Liebmann Fellowship & NIH Grant ES05511
Structure and regulation of gpx2 gene expression by selenium in mouse intestine and colon
Fong-Fong Chu*, R. Steven Esworthy*, Weiya Wang¶, Leslie Lee*, and Ye-Shih HoÝ
*Department of Medical Oncology, ¶Department of Neuroscience, City of Hope Beckman Research Center, Duarte, CA 91010, and ÝInstitute of Toxicology, Wayne State University, Detroit, MI 48201
We have isolated a mouse gpx2 gene encoding GPX-GI from a P1 library constructed from 129/Sv embryonic stem cells. Similar to Gpx1 gene, which encodes the classic GPX-1, Gpx2 also has two exons and one intron. We have since obtained 6.5kb sequence including the two exons, 2.2kb intron, 2kb of 5¹ untranslated region, and 1kb of 3¹ untranslated region from this gene. Since Gpx2 is highly expressed in the GI-tract, and GPX-GI is a selenoprotein, we have analyzed the effect of selenium on gpx2 gene expression at the mRNA and protein levels in the GI-tract of mice with disrupted Gpx1 gene, i.e. Gpx1-KO, and of control mice. Unlike Gpx1 gene expression which is highly responsive to sele