R. Stocker, A.R. Waldeck, P.K. Witting, K.E. RyeÝ, J.K. Christison, M. Raftery¶, and B. Garner
Biochemistry and ¶Immunology Groups, The Heart Research Institute, Sydney, and ÝRoyal Adelaide Hospital, Adelaide, Australia
Oxidation of high density lipoproteins (HDL) can decrease the anti atherogenic activities of these lipoproteins, and in human atherosclerotic plaque the lipids in apolipoproteinAI- (apoAI) and apoAII-containing high density fraction are as oxidized as those in the apoB-containing low density fraction. Here we report on the relative susceptibility of HDL lipid and protein to oxidation and on the role HDL's a tocopherol (a TOH) plays in modulating protein oxidation. Exposure of HDL to either low fluxes of peroxyl radicals (ROO€), Cu(II), or soybean lipoxygenase (SLO) resulted in the oxidation of apoAI and apoAII after consumption of ubiquinol-10 but in the presence of a-TOH. Cholesteryl ester (CE-OOH) and phospholipid hydroperoxides (PC OOH) initially accumulated together with the corresponding hydroxides (i.e., CE-OH and PC-OH), as well as specific oxidized forms of apoAI and apoAII that contained Met sulfoxide (Met(O)) instead of Met at selective residues as the single modification. Exposure of isolated apoAI to ROO€, Cu(II), or SLO resulted in non-specific or no protein oxidation, whereas treatment of apoAI with authentic CE-OOH gave rise to specifically oxidized apoAI and CE-OH. Enrichment of HDL with a TOH increased the oxidant-induced formation of oxidized apoAI/AII and CE-OOH/CE-OH. The observed reducing activity of native and a TOH-enriched HDL for lipid hydroperoxides (LOOH) was similar in HDL2 and HDL3, independent of aryl esterase and LCAT activity, unaffected by sulfhydryl reagents, and was expressed by reconstituted particles containing human apoAI or apoAII only. As LOOH became reduced, the specifically oxidized apoAI and apoAII accumulated. Reduction of LOOH to LOH and formation of oxidized apoAI and apoAII were inhibited by solubilizing HDL with SDS or by pre-treating HDL with chloramine-T, which converted Met of apoAI/AII into Met(O).
Canine HDL, which contains apoAI as the predominant apolipo protein but lacks the oxidation-sensitive Met residues Met112 and Met148, showed much weaker LOOH reducing activity and lower extents of formation of oxidized apoAI than human HDL. We conclude that the oxidation of specific Met residues of apoAI and apoAII to Met(O) plays a significant role in the two-electron reduction of CE-OOH and PC-OOH associated with human HDL, and that this is an early event in HDL oxidation that can be modulated by vitamin E.
The effects of ascorbic acid deficiency on the human aorta endothelial cells
Nozomu Tamai and Takao Fujinami
Internal Medicine, Nagoya City University Medical School, Nagoya, Japan
Background: In the scurvy prone Osteodystrophy Shionogi (ODS) rats, electron microscopic observations revealed that the aortic and coronary endothelial cells of the ODS rats were flattened and lost the undulation of the surface, and pinocytic vesicle in the lumen site cytoplasm was decreased. The basement membrane of the endothelium got thinner. In the aorta, there observed monocytes/macrophages adhered on the endothelial surface and invaded into the subendothelial space.
Objective: To evaluate the mechanisms of the protective effects of ascorbic acid (AsA) on the vascular endothelial cells from oxidative stresses and its involvement in the LDL peroxidation.
Methods: 1) 8 weeks old, male ODS (od/od) rats were fed with AsA deficient diet for 3 weeks. Then the serum was separated, and incubated for 30 minutes at 56°C for inactivation of complements. This serum was added to the cell growing medium (HuMedia EG2) at a concentration of 5% (AsA deficient medium). 20 mg/dl of AsA was added to AsA deficient medium (AsA sufficient medium). Human aortic endothelial cells (HAECs) were cultured to 3,000 /cm2 or to confluent. Then the mediums were changed to AsA deficient or sufficient medium. Cell morphology and density were estimated on the 2, 4, and 7th days. 2) Human LDL isolated from pooled sera of healthy normolipidemic individuals was incubated for 6 hrs with 2.5µM Cu2+ in PBS (ox-LDL), or in the presence of additional 20 mg/dl AsA (AsA treated LDL). HAECs were cultured in the same way, and the mediums were changed to those containing native LDL, oxLDL, AsA treated LDL, PBS. The morphological change and the density were observed on the 2, 4, 7th day.
Results: 1) HAECs grown in the AsA sufficient medium proliferated to 14,592 ± 1,563 /cm2 from 3,000 /cm2, decreased to 11,872 ± 1,005 /cm2 from confluent (15,693 ± 1,226 /cm2) in 7 days, whereas those grown in AsA deficient medium proliferated to 7,008 ± 387 /cm2, and decreased to 5,472 ± 387 /cm2 from confluent (p<0.01 for both). 2) HAECs grown in the medium containing NLDL as well as that of PBS was normal endothelial shape, and proliferated to 11,820 ± 178 /cm2 from 3,000 /cm2, and decreased to 7,877 ± 966 /cm2 from confluent in 7 days. But those grown in ox-LDL conditioned medium were spindle shaped and exhibit growth inhibition to 8,255 ± 559 /cm2 from 3,000 /cm2, and decrease in number to 1,913 ± 164 /cm2 from confluent (p<0.01). On the contrary, cells grown in the medium containing AsA treated LDL showed no significant difference in number (11,387 ± 376 /cm2 from 3,000 /cm2 and 9,186 ± 1,002 /cm2 from confluent) nor morphological change from those containing NLDL or PBS.
Conclusions: AsA protected HAECs from oxidative damages. One of the mechanism was suggested to be involved in the inhibition of the peroxidization of LDL. The evidence indicates that ascorbic acid is playing an important role in the prevention of endothelial injury by ox-LDL.
Generation of a-tocopherol transfer protein knockout mice
Yuko Terasawa, Sylvaine Cases, Maret G. Traber, Lester Packer, and Robert V. Farese, Jr.
University of California, Berkeley and San Francisco
Vitamin E is a potent antioxidant transported through the circulation in plasma lipoproteins to be further distributed to tissues. a-tocopherol transfer protein (a-TTP) is a cytosolic protein expressed in hepatocytes that is thought to enrich plasma lipoproteins and tissues in thea-tocopherol form of vitamin E. Genetic defects of a-TTP in humans result in the syndrome of ataxia with vitamin E deficiency (AVED), a rare disorder in which low plasma a-tocopherol levels result in neurologic disease. In preliminary studies, we found that a-TTP is also expressed in the mouse yolk sac, a tissue that plays a crucial role in maternal-fetal transport of nutrients in rodents.
To study the in vivo role of a-TTP in mouse embryonic development and to generate a genetically induced model of vitamin E deficiency, we have disrupted the a-TTP gene (Ttp) in mice. A gene targeting vector was designed to delete the Ttp translational start codon and to insert lacZ into the Ttp locus under the control of the Ttp promoter. The latter feature will provide us with a sensitive technique to analyze Ttp expression in cells of embryos and adult mice. Electroporation of the targeting vector into RF8 embryonic stem (ES) cells resulted in the isolation of 4 (of 833) targeted ES cell clones; one of these targeted ES cell clones was subsequently used to generate chimeric mice that transmitted the knockout mutation through the germline. We are currently intercrossing Ttp+/- mice to determine the phenotype of complete a-TTP deficiency in mice. a-TTP knockout mice will enable us to test whether a-TTP is essential for mouse development and will provide an animal model to investigate the mechanism whereby a-TTP enriches plasma lipoproteins in a-tocopherol. a-TTP deficient mice will also provide a mouse genetic model of vitamin E-deficiency to investigate the role of this antioxidant in processes such as neurologic disease and atherosclerosis.
S.E. Terblanche, T.C. Masondo, and W. Nel
Department of Biochemistry and Microbiology, University of Zululand, Private Bag X1001, Kwa-Dlangezwa 3886, South Africa
The effects of cold acclimation on lactate dehydrogenase and its isoenzymes in various tissues of the rat (Rattus norvegicus) were investigated. Male Sprague-Dawley rats were divided into two groups. The one group was housed at 4 ± 1°C (experimental group) and the other at 24 ± 1°C (control group) for six mongths. The rats were housed insingle cages and had access to food and water ad libitum.
On the day of sacrifice, rats were anaesthetised by an intraperitoneal injection of pentobarbitone (6 mg/100 g body mass). Blood was drawn from the abdominal aorta into a heparinised syringe and immediately centrifuged to obtain serum. The heart, liver, lung, kidnez, and gastrocnemius muscle were excised and camp frozen with tongs cooled in liquid air. Interscapular brown adipose tissue were also excised. All samples were kept in liquid air prior to the determination of the activity levels of lactate dehydrogenase. Isoenzymes of lactate dehydrogenase were separated by electrophoresis and quantified. All results were analysed statistically using students t-test (two tailed).
Total lactate dehydrogenase activity levels were significantly increased (p < 0.05) in all the tissues investigated as well as serum. The HM3 isomer was significantly elevated in all the tissues investigated as a result of cold acclimation. Cold acclimation resulted in an increase in all detectable isoenzymes, although not always significant, in all the tissues investigated as well as serum. The M4 isomer was the only one detectable in serum. In general, the results are indicative of a compensatory adaptation to cold oxidative stress evaluated on the response of lactate dehydrogenase and its isoenzymes.
Glutamateinduced apoptosis in HT-4 neuronal cells: Mechanisms and the role of antioxidants
Oren Tirosh, Chandan K. Sen, Sashwati Roy, Michael S. Kobayashi, and Lester Packer
Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200
Elevated levels of extracellular glutamate has been linked to neuronal damage and brain disorders including stroke, epilepsy, and Parkinson¹s disease This study was aimed to clarify the involvement of oxidants and the role of antioxidants in glutamate induced cytotoxicity in mouse hippocampal HT-4 cells. Glutamate treatment (10 mM) for 8 h resulted in the onset of apoptosis as observed by changes in DNA integrity. Almost all cells lost viability 12 h after treatment. Flow cytometric measurement of intracellular peroxides in glutamate treated cells using the probe dihydrochlorofluorescein (DCFH-DA) show ed marked (200%-250%) increase following 6 h of glutamate treatment. Treatment of cells with various thiol and phenolic antioxidants protected against such intracellular peroxide accumulation and subsequent cell death. Lipophilic antioxidants were more effective in this respect. a-Tocopherol and methyl-trolox protected the cells at concentrations as low as 1 µM and 2 µM respectively. In contrast, the water soluble analogue of tocopherol, Trolox did not protect. The Nitroxides, Tempo and Tempol, a different class of antioxidant were also tested. Lipophilic Tempo protected much more effectively than hydrophilic Tempol. a-Lipoic acid also protected the cells. The protective function of this water soluble thiol antioxidant synergised with pre-treatment of the cells with 1 µM sodium selenite 24 hours prior to glutamate challenge. Flow cytometric measurement of the red and green fluorescence of the ratio-metric dye JC-1 showed that glutamate induced marked increases in mitochondrial mass. Western blots showed increased levels of Bcl-2 in glutamate treated cells. Both the mitochondrial and Bcl-2 accumulation were found to be regulated by antioxidants. Results of this study indicated that lipophilic antioxidants are particularly effective in preventing glutamate induced cell damage, and that glutamate dependent changes in mitochondria function may contribute to glutamate induced apoptosis.
*Hitoshi Togashi, *Haruhide Shinzawa, §Tateaki Ogata, *Taku Matsuo, *Shigeru Ohno, *Koji Saito, *Nobuo Yamada, ¶Hidekatsu Yokoyama, ¶Hiroyuki Noda, ÝKazuo Oikawa, ¶Hitoshi Kamada, and *Tsuneo Takahashi
*The Second Department of Internal Medicine, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-23, §Department of Materials Science and Engineering, Faculty of Engineering, Yamagata University, Yonezawa 992, ¶Institute for Life Support Technology, Yamagata Technopolis Foundation, 2-2-1 Matsuei, Yamagata 990, ÝYamagata Research Institute of Technology, 2-2-1 Matsuei, Yamagata 990, Japan
Electron spin resonance (ESR) imaging can visualize the distribution of free radicals in living systems according to their concentrations. However, the application of ESR imaging to living animals has not been well established. Using a rapid field scan L-band ESR imaging system, we have successfully obtained two-dimensional ESR projection (XZ-plane projection) and three-dimensional ESR-CT (trans- axial section along the Y-axis) images of the abdomen of living mice after an injection of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1- oxyl (carbamoyl-PROXYL) into the tail vein. The in vivo two-dimen sional ESR projection imaging clearly visualized the carbamoyl- PROXYL distribution and the rapid decay process in the abdomen. Because among the viscera, the liver is most abundantly associated with a blood volume, the outline of the image can be composed mainly of this organ. We therefore attempted to find whether there will be a difference in spatiotemporal dynamics of carbamoyl- PROXYL in the abdomens between the control and the mice with liver damage by two-dimensional ESR projection. In the control mice, carbamoyl- PROXYL was almost completely eliminated from the ab-domen within 5 minutes after administration. On the other hand, in mice with carbon tetrachloride-damaged livers, the decay of carbamoyl-PROXYL was markedly prolonged. Even at 5 minutes after administration, carba moyl-PROXYL remained clearly visible in the abdomen. In vivo three-dimensional ESR-CT imaging showed an even distribution of carbamoyl-PROXYL throughout the whole liver, which corresponded well with the images of trans-axial sections of the murine abdomen. We have succeeded in displaying two-dimensional ESR projection and three-dimensional ESR-CT images of carbamoyl-PROXYL distribution and clearance in the abdomen of a living animal. The ESR-CT imaging technique is considered to be a powerful new tool for noninvasive investigations of the in vivo spatio-temporal dynamics of free radical distribution and elimination in the organs.
J.R. Trevithick, V. Bantseev, and T. Dzialoszynski
Department of Biochemistry, Faculty of Medicine, The University of Western Ontario, London, Canada
Aims: Previous experiments had indicated that 1 mM R-a-lipoate(RLA) protected lenses against damage in model diabetic cataract, but caused fragmentation of elongated fiber cell mitochondria after as little as 6 hr incubation. These experiments were performed to test whether a lower concentration of RLA (10 µM) in the incubation medium could reduce mitochondrial fragmentation and still protect the lens against model diabetes (55.6 mM glucose). Methods: Rat lenses were incubated in 55.6 mM glucose in medium 199 (M199) after a 24 hour preincubation to check for possible damage by 1) opacity, 2) protein leakage. Any lenses exhibiting damage were discarded. Lenses were stained with 14 µM rhodamine-123 for 15 minutes to stain mitochondria, immobilized in 1% agarose in M199, and the equatorial region examined by a Zeiss confocal microscope using a water-immersion lens, after 6 hrs and 8 days using as negative control CCCP (65 µM), an electron transport inhibitor. Results: Initially and in control lenses, epithelial mitochondria staining was punctate, while in fiber cells long sinuous mitochondria could be seen. After 6 hr. fiber cells of control lenses and lenses incubated in 10 µM RLA still had long sinuous mitochondria, while in 1 mM RLA mitochondria showed punctate staining. After 8 days in 55.6 mM glucose the controls (M199 or 0.1% ethanol) showed few fiber cell mitochondria, all punctate. In 1 mM RLA punctate mitochondria were seen, although fewer than at 6 hr. In 10 µM RLA many fibrous mitochondria were seen. Conclusions: Although mitochondrial fragmentation occurs along with cataract risk reduction in 1 mM RLA, 10 µM RLA appears additionally to protect mitochondrial structure and function in the in vitro diabetic lens model.
Supported by Defence Research Establishment, Ottawa and , Henkel, Fine Chemicals Div., Asta Medica, Frankfurt, and Eastman Fine Chemicals.
T. Tsujita,2, S. Okamoto2, K. Tajima3, K. N. Azuma1, M. Kohno4 K. Makino5, and K. Ishizu1
1Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790; 2Coating Division, NOF Corporation, Totsuka-ku, Yokohama 244; 3Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku Kyoto 606; 4Application Center, Analytical Division, JEOL Ltd., Akishima, Tokyo 196; 5Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611, Japan
Shikonin has been well established as one of the noble ancient purple dye, so called shikon, which was purified from a root of lithospermumerythrorhizon. This dye was consisted of a naphtazarine and alcoholic moieties, and was believed to have noble anti-oxidant activity against active oxygen species. However, detailed antioxidant activity of the dye was still equivocal, in spite of the wide spread usage of the dye to a recent functioned cosmetics. We have explore the redox behavior of the dye in relation to the scavenging activity for superoxide and hydroxyl radicals, generated in inorganic and biological systems. The mechanisms of the reaction between the dye and these radicals will be discussed based on the results of ESR, ENDOR and CV measurements.
Reactive oxygen species and nitric oxide production by human blood mononuclear cells
Laura Valdez, Silvia Lores Arnaiz, and Alberto Boveris
Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina
The reduction products of oxygen, such as superoxide anion (O2-) and hydrogen peroxide (H2O2), are generated as the result of the respiratory burst by polymorphonuclear leukocytes (PMN) and monocytes. NADPH oxidase catalyzes the reduction of oxygen to O2- using NADPH. This enzyme was found in phagocytes and B lymphocytes, though the role of O2- production in B lymphocytes has not been extensively studied. On the other hand, it has been shown that human neutrophils produce nitric oxide (.NO) by activation of nitric oxide synthase (NOS) when they are exposed to appropriate stimuli. The presence of NOS has not been reported in non-phagocytic blood cells. In this work we studied the activity of NADPH oxidase and the .NO production in isolated human mononuclear cells (MNC). The response observed can be attributed to the lymphocytes because these cells constitute the highest proportion in the mononuclear fraction (90%).
Mononuclear cells were isolated by Ficoll-Hypaque gradient separation from blood of healthy human donors. Luminol-amplified chemiluminescence (CL), oxygen consumption, and the production of O2-, H2O2 and .NO were determined at 37šC, before and after stimulation with 0.1 g/ml phorbol 12-myristate 13-acetate (PMA).
PMA-stimulated cells showed a 13-fold increase in CL and 2-fold increase in oxygen consumption. Production of O2- was 0.59 ± 0.19 nmol O2-/min.106 cells in activated cells (resting cells value: 0.08 ± 0.01 nmol O2-/min.106 cells). The steady-state concentration of H2O2 increased 2-fold in PMA treatment. Production of .NO measured by oxyhemoglobin method was increased when MNC were stimulated with PMA, in the presence of calcium. In addition, .NO signal was detected by EPR/spin trapping technique.
The physiological significance of O2- and .NO production in lymphocytes is still not clear. Possible functions may include participation in antigen processing and intracellular signaling. Further studies are necessary to elucidate the role of these species in activated lymphocytes.
The physical state of membranes affects the rate of lipid oxidation. Effect of metals
Sandra V. Verstraeten and Patricia I. Oteiza
Departamento de Química Biológica, IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina
We have previously described that aluminum (Al3+) and other related non-redox metals stimulate lipid oxidation (evaluated as TBARS, conjugated dienes and fatty acids concentration) in the presence of Fe2+ and that this effect was correlated with the capacity of the metals to promote fusion, aggregation and permeabilization of membranes. We have further characterized the mechanism through which metals whithout redox capacity (Al3+, Ga3+, Sc3+, Y3+, In3+, La3+, Be2+) can stimulate lipid oxidation in the presence of an initiator (Fe2+) in liposomes and brain membranes.
Results showed that: a) The ability of the ions to alter membrane lipid packing and induce lateral phase separation in liposomes correlated in a positive manner (r2 = 0.91 and 0.90 respectively) with their capacity to stimulate the production of TBARS, b) when compared to mitochondria, microsomal and synaptic membranes, myelin showed a marked susceptibility (3.5, 2.1 and 2.2-fold higher respectively) to Al3+-mediated lipid oxidation. Results indicate that this is due to the high relative content lipid:protein of myelin compared to other membranes, c) the presence of galactolipids in liposomes caused a concentration (10-40 mol % galactolipids)-dependent increase in the stimulatory effect of Al3+ on Fe2+-induced TBARS production. This was associated with an increase in the capacity of Al3+ to induce lateral phase separation (r2 = 0.99) and rigidification.
The present results support the hypothesis that factors (metals, galactolipis, membrane composition) that increase lipid packing and promote the formation of rigid clusters of phospholipids in membranes will favor lipid oxidation
Supported by grants from the Universidad de Buenos Aires (FA116, FA055), CONICET and Fundación Antorchas