Biochemistry of oxidative stress

39th Meeting of the Polish Biochemical Society Gdañsk 16–20 September 2003 SESSION 1 Biochemistry of oxidative stress Organized by M. WoŸniak, G. B...
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39th Meeting of the Polish Biochemical Society Gdañsk 16–20 September 2003

SESSION 1

Biochemistry of oxidative stress

Organized by M. WoŸniak, G. Bartosz, T. Wakabayashi

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Session 1. Biochemistry of oxidative stress

2003 Lecture

3 The role of a-tocopherol in preventing disease Angelo Azzi Institut für Biochemie und Molekularbiologie, Universität Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland

A role of oxidative stress in atherosclerosis lies on experimental results carried out in vitro and in animal models. In humans, the supplementation with the antioxidant vitamin E has given in some cases supportive results and in others no effects. From in vitro studies, a large amount of data has shown that a-tocopherol (the major component of vitamin E) regulates key events in the cellular pathogenesis of atherosclerosis. We have first described the inhibition of protein kinase C (PKC) activity by a-tocopherol to be at the basis of the vascular smooth muscle cell growth inhibition by this compound. Subsequently, PKC was recognized to be the target of a-tocopherol in different cell types, including monocytes, macrophages, neutrophils, fibroblasts and mesangial cells. Inhibiting the activity of protein kinase C by a-tocopherol results in different events in different cell types: inhibition of platelet aggregation, of ni-

tric oxide production in endothelial cells, of superoxide production in neutrophils and macrophages as well as impairment of smooth muscle cell proliferation. Adhesion molecule expression and inflammatory cell cytokine production are also influenced by a-tocopherol. Scavenger receptors, particularly important in the formation of atherosclerotic foam cells, are also modulated by a-tocopherol. The oxidized LDL scavenger receptors SR-A and CD36 are down regulated at transcriptional level by a-tocopherol. The relevance of CD36 expression in the onset of atherosclerosis has been indicated by the protection against atherosclerosis by CD36 knockout mice. In conclusion, the effect of a-tocopherol against atherosclerosis is not due only to the prevention of LDL oxidation but also to the down regulation of the scavenger receptor CD36 and to the inhibition of PKC activity.

Lecture

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Stimulation of radical formation accompanying metabolic activation of adriamycin by cytochrome P450 reductase — friend or foe? 1

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Agnieszka Bartoszek , Zofia Mazerska , Jolanta Paw³owska , Grzegorz Bartosz , Mark Paine , Roland Wolf

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1 — Department of Pharmaceutical Technology and Biochemistry, Gdañsk University of Technology, Gdañsk, 2 — Katedra Biofizyki Molekularnej, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ, 3 — Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom

Previous studies demonstrated that metabolic activation of adriamycin by NADPH cytochrome P450 reductase (P450red) potentiated cytotoxicity of this drug towards cultured tumour cells. This observation suggested that one-electron reduction might play an essential role in antitumour activity of anthracyclines. Since the metabolic convertion by P450red is associated with the sequential changes of adriamycin UV-VIS spectrum, we were able to monitor this drug reduction and to analyse spectra of intermediates. The application of spectrophotometric method in combination with EPR measurements made it possible to relate the appearance of particular adriamycin metabolites to the generation of superoxide radicals formed as a result of reaction of adriamycin semiquinone radicals with molecular oxygen. Based on results obtained and the data available in literature, we proposed the route of adriamycin metabolic conversion by P450red. We suggest, that this process involves five stages, two of which are accompanied by intensive redox cycling (stages 2 and 3).

We also studied the interaction of P450 reductase-generated adriamycin metabolites with nucleic acids in cell-free system. The assumption was that the formation od adriamycin-DNA adducts should in a way preserve the structure of the active metabolite(s) which will be reflected by spectral properties of reaction mixture. DNA and oligodeoxynucleotides blocked adriamycin reduction at the stage 3 of metabolism corresponding to the tautomerization of the leuko form of adriamycin to hydroquinone. The formation of adriamycin species capable of covalent bonding with DNA during stage 3 of its enzymatic reduction by P450red was confirmed by the newly developed in our laboratory method exploiting restriction enzymes to the detection of covalent DNA modification. So far, the stimulation of redox cycling by antitumour anthracyclines has been regarded mainly as a reason of toxic side effects, cardiotoxicity in particular. Our findings made us look upon this process quite differently. The formation of DNA-binding adriamycin metabolite seems to take place at the same stage of metabolism, at

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39th Meeting of the Polish Biochemical Society

which intensive redox cycling occurs. It follows that in tumour cells, this active metabolite may proceed via three routes: it may reach its target (DNA), it may be further reduced to inactive species or it may react with

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molecular oxygen thereby returning to its previous form liable to the next round of activation (producing toxic oxygen species at the same time).

Lecture

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Oxygen-dependent and oxygen-independent effects of superoxide dismutase deficiency in yeast 1

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Tomasz Biliñski , Jaros³aw Wawryn , Anna Krzepi³ko , Agata Œwiêci³o , Renata Zadr¹g , Ewa ¯yracka , 1 Grzegorz Bartosz 1 — Instytut Biologii i Ochrony Œrodowiska, Uniwersytet Rzeszowski, ul. Rejtana 16C, 35-310 Rzeszów, 2 — Instytut Nauk Rolniczych w Zamoœciu, Akademia Rolnicza w Lublinie, Zamoœæ

Since the discovery of enzymatic activity of erythrocuprein, the physiological role of superoxide dismutase (SOD) was a center of heated debate. The in vivo importance of disproportionation of superoxide anion by SOD was proved by isolating the sod1 mutants of yeast, deficient in CuZnSOD which appeared to be hypersensitive to oxidative stress. However, not all characteristic features of the sod1 mutant cells are oxygen dependent. Deficiency in CuZnSOD shortens generative life span of the mutant cells and results in conditional lysine and methionine auxotrophy. Nutritional requirements connected with sod1 mutation are clearly oxygen dependent because lysine auxotrophy is not ob-

served at oxygen concentrations below 1%, whereas methionine (reduced sulphur) requirement is observed below 5% of O2. In addition, these defects can be compensated by a typical antioxidant — ascorbate. In contrast, the influence of oxygen on the life span of the mutant cells is negligible in the range of concentrations between 0 and 21%. Supplementation of the medium with ascorbate substantially extends life span of the mutant cells, but only under aerobic conditions. These results suggest that not all sequelae of superoxide dismutase deficiency can be ascribed to the classical main function of this enzyme.

Lecture

6 Regulation of telomerase activity by glutathione levels Consuelo Borras, Juan Esteve, Dolores Royo, Luis Torres, Juan Sastre, Jose Vina, Federico Pallardo Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain

The eukaryotic chromosomes are capped by the telomeres, which are tandemly repeated telomeric DNA, associated with several proteins. These structures play an important role in the stability and the complete replication of the chromosomes. Conventional DNA polymerases cannot fully replicate the 3’ end of the lagging strand of linear molecules and therefore, in every cell division, telomeric sequences are lost. This telomere shortening works as a molecular clock, because after a certain point of divisions and telomere loss, the cells are no longer capable of dividing and they enter a state called senescence or aging. One way of resetting this molecular clock and restoring the telomere length is by telomerase, a ribonucleoprotein complex that synthesizes telomeric DNA de novo and also caps the ends of telomeres, thus contributing to chromosome stability.

Telomerase regulation is multifactorial and not well understood, but changes in it are related to both cancers and aging. The aim of our study was to determine the possible role of glutathione in the regulation of the telomerase activity. Our results show a positive correlation between GSH levels and telomerase activity in 3T3 fibroblasts and fibroblasts from human foreskin. Cells reached confluency after a week in culture. The peak of telomerase activity coincides with GSH peak. Depletion of GSH with buthionine sulphoximine (BSO) reduces the telomerase activity after 24 and 48 hours of treatment. BSO treated cells incubated with GSH ester restores both GSH levels and telomerase activity. We present results showing the role of E2F and pRb in the regulation of telomerase activity by GSH.

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The reported changes during the time of culture also correlate with changes in the percentage of cell in the different stages of cell cycle G1 and S+G2/M.

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We can conclude that GSH levels in cells could regulate, at least in part, telomerase activity.

Lecture

7 Inhibition of basal and IL-1-induced VCAM-1 expression by PHGPx and 15-lipoxygenase in rabbit aortic smooth muscle cells Regina Brigelius-Flohe, Antje Banning, Kerstin Schnurr

German Institute of Human Nutrition, Potsdam-Rehbruecke, Institute of Nutritional Science of the University of Potsdam, Arthur-Scheunert Allee 114-116, Bergholz-Rehbruecke, Germany

The oxidation hypothesis is one of the central hypotheses to explain the process of atherogenesis. It includes the oxidative modification of LDL and other lipoproteins, which by themselves or by their various oxidation products including fatty acid hydroperoxides initiate the atherosclerotic process. At early stages of atherosclerosis, endothelial and smooth muscle cells upregulate cell adhesion molecules (CAM). CAM-expression can be upregulated by cytokines such as interleukin-1 (IL-1) or by hydroperoxides, which may be products of LDL oxidation or intermediates of IL-1 signalling. We therefore investigated whether the cellular hydroperoxide tone modified by phospholipid hydroperoxide glutathione peroxidase (PHGPx) or 15-lipoxygenase (15-LOX) influences basal and IL-1-mediated VCAM-1 expression. We made use of a model of rabbit aortic smooth muscle cells (SMC) stably transfected either with PHGPx (SMCPHGPx) as a hydroperoxide-reducing or 15-LOX (SMCLOX) as a hydroperoxide-producing enzyme. Characterization of the cells revealed successful transfection by an enhanced activity of the respective enzyme. Intracellular hydroperoxides were 6-fold higher in SMCLOX than in SMC or SMCPHGPx. Cel-

lular protein thiols were decreased by 50% and 90% in SMCPHGPx and SMCLOX, respectively. Thiol reduction with Tris(2-carboxyethyl)-phosphine completely restored protein thiols in SMCPHGPx whereas in SMCLOX thiols reached about 50% of control values. Compared to wild type SMC, basal VCAM-1 mRNA levels were decreased by 50% in SMCPHGPx, an effect which correlated with selenium-dependent enzyme activity. In SMCLOX the decrease in basal VCAM-1 expression was even more pronounced. Whereas the inducibility of VCAM-1 by IL-1 was maintained, but delayed, in SMCPHGPx, it was abrogated in SMCLOX. RNA data were confirmed by immunocytochemistry. PHGPx effects support the view that a higher peroxide tone facilitates IL-1 signalling and in consequence VCAM-1 expression. Alternatively, PHGPx may dampen CAM expression by making use of hydroperoxides for protein modification. Overproduction of hydroperoxides as observed in SMCLOX can also prevent CAM expression via oxidation of protein thiols and make cells refractory for further stimuli. In summary, any disturbance in the cellular redox state impairs the expression of vascular cell adhesion molecules in vascular smooth muscle cells.

Lecture

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Single- and two-electron reduction of quinones by flavoenzymes: mechanisms and implications for quinone cytotoxicity 1

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Narimantas Cenas , Ausra Nemeikaite-Ceniene , Jonas Sarlauskas , Juzefa Acaite

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1 — Biochemistry, Institute of Biochemistry, Mokslininku St. 12, Vilnius, Lithuania, 2 — Immunology, Institute of Immunology, Moletu Pl. 29, Vilnius, Lithuania

For manifestation of their therapeutic and/or toxic properties, most quinones have to undergo an enzymatic single-electron reduction (e.g., by NADPH:cytochrome + P-450 reductase (P-450R), ferredoxin:NADP reductase (FNR)), or two-electron reduction by DT-diaphorase (NQO1). We examined the reactivity of the above flavoenzymes with aziridinyl-substituted quinones (AZQ, DZQ, MeDZQ, RH1, BZQ), daunorubicin, natural

hydroxyanthraquinone pigments, and model quinones (n = 38). Quinone reactivity (log kcat/Km) in reactions with P-450R and FNR exhibited parabolic dependence on their single-electron reduction potential (E17), slightly increased with the number of their aromatic rings (N = 1–3, FNR), or was increased for naphthoquinone structure (N = 2, P-450R), and did not depend on their Van der Waals volume (VdWvol), or pKa of

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39th Meeting of the Polish Biochemical Society

their semiquinones (pKa(QH.). In reactions of NQO1, log kcat/Km of quinones exhibited the parabolic dependence on their E17, but also showed positive dependence on pKa(QH.), and a strong negative dependence on VdWvol. Alternatively, an equal specificity for benzoand naphthoquinone structures (N=1–2) with a negative reactivity dependence on VdWvol of substituents may be demonstrated. The reaction activation entropies increase with an increase in the VdWvol of quinones, im3 plying that VdWvol < 200 Å is optimal for an efficient electron coupling during two-elctron reduction of quinones by NQO1. In FLK cell line, the toxicity of aziridinyl-substituted quinones was higher than that of of aziridinyl-unsubstituted ones. Both compound series showed a negative log cL50 dependence on their E17. The toxicity

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of anthraquinone pigments with unavailable E17 in FLK and HL-60 cells correlated with the rates of their enzymatic redox cycling. The potentiating effects of BCNU, protecting effects of antioxidants and desferrioxamine, and lipid peroxidation show that quinone cytotoxicity is manifested through their redox cycling and oxidative stress. The toxicity of aziridinyl-substituted quinones was party prevented by dicumarol, which shows an additional activation by NQO1. The toxicity of daunorubicin in FLK and HL-60 cell lines was also higher than expected from its redox cycling rate, although being decreased by antioxidants and desferrioxamine. This discrepancy was caused by an enhanced cellular accumulation of daunorubicin, which was not characteristic for hydroxyanthraquinone pigments.

Lecture

9 Iron-mediated stress signalling Herbert De Groot Institut fuer Physiologische Chemie, Universitaetsklinikum, Hufelandstrasse 55, Essen, Germany

Iron ions are essentially involved in a great variety of cell and tissue injurious processes. This concerns not only iron-storage diseases but also other injuries such as reperfusion injuries and neurodegenerative diseases like Parkinson’s and Alzheimer’s diseases. The iron ions involved belong to an iron pool that is usually defined as labile or chelatable iron or as iron in transit. They are loosely bound to various constituents of the cells, small and large ones, such as ADP, citrate, proteins and lipids. They are redox-active and thus their redox state may cycle between the ferrous [Fe(II)] and the ferric state [Fe(III)]. Due to their characteristics, the iron ions of the labile iron pool are difficult to determine. Recently fluorescent microsopic methods have been developed that allow the determination of these iron ions with even subcellular resolution within viable cells.

Due to their redox properties the iron ions of the labile iron pool are capable of catalysing the activation of hydrogen peroxide (and other peroxides) and of molecular oxygen to true oxidants such as the hydroxyl radical. This capability is the basis for their contribution to injurious processes. A fairly well studied example of such a process is cold-induced apoptosis which may decisively contribute to the storage injury of an organ during transplantation. It is initiated by an increase in the concentration of the iron ions of the labile iron pool occurring during the cold period. Upon rewarming, e.g. upon reperfusion following transplantation, apoptotic processes are induced. Mitochondrial alterations appear to be decisively involved in these processes. Antioxidants such as appropriate iron chelators in conjunction with additional measures aimed to protect from hypoxic cell injury can largely improve the quality of the stored organs.

Lecture

10 The changing face of glutathione, a cellular evergreen 1

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Silvia Dominici , Lisa Pieri , Maria Franzini , Aldo Paolicchi , Alfonso Pompella

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1 — Dept. of Pathophysiology and Exp. Medicine, Univ. of Siena, Siena, Italy, 2 — Dept. of Experimental Pathology B.M.I.E., University of Pisa, Pisa, Italy, 3 — Dept. of Experimental Pathology, Scuola Medica, University of Pisa, via Roma 55, I-56126 Pisa, Italy

A number of biomolecules involved in crucial cellular functions, such as signal transduction and regulation of

gene expression, are sensitive to prooxidants. The latter are active in this case at concentrations considerably

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lower than those capable to induce oxidative injury. Prooxidants thus can no longer be regarded as merely offensive species, and similarly, the physiological role of some established ‘antioxidants’ also is in need of careful reconsideration. Glutathione — perhaps the best known cellular ‘antioxidant’ — appears as an ideal candidate. The antioxidant role of GSH is readily apparent in detoxification of electrophilic/oxidizing drugs and protection from lipid peroxidation. In these processes most of GSH action is achieved through the action of GSH-dependent enzymes, GSH-S-transferases and GSH-peroxidases. Non-antioxidant functions of GSH have been described e.g. in modulation of signal transduction, cell proliferation and immune response. The ability of GSH to affect the thiol redox status of critical proteins (receptors,

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transducers, transcription factors) plays a major role in this kind of processes. To complete the picture, recent studies point to prooxidant effects of (extracellular) GSH, which can ensue from its catabolism by the membrane ecto-enzyme gamma-glutamyltransferase (GGT). It has in fact been documented in our and other laboratories that prooxidant species (superoxide, H2O2, thiyl radicals) are produced during GSH catabolism, as a result of the interaction of GSH metabolites — glycyl-cysteine in the first place — with trace levels of redox-active metal ions present in the cell environment. The significance of GSH and reactions related to its transport and metabolism — after decades of fruitful research — still holds aspects to investigate and plots to unveil.

Lecture

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New copper (I) complexes protect against DNA damage in rat epithelial cells induced by nitric oxide, nitroxyl anion and peroxynitrite 1

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Donatella Fedeli , Giancarlo Falcioni , Carlo Santini , Maura Pellei , Giancarlo Gioia Lobbia

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1 — Department of MCA Biology, University of Camerino, Camerino, Italy, 2 — Department of Chemistry, University of Camerino, Camerino, Italy

In order to gain more knowledge on the antioxidant role of copper(I) complexes, in this study we investigate their possible protective action against DNA damage induced by nitric oxide (NO) and reactive nitrogen oxide species deriving from it, namely nitroxyl anion – – (NO ) and peroxynitrite (ONOO ). Rat trachea epithelial cells were exposed under aerobic conditions to (i) NO generated by 150 mM S-nitrosoglutathione – monoethyl ester (GSNO-MEE), (ii) NO generated by – 200 mM Angeli’s salt (Na2N2O3) (iii) ONOO generated by 1 mM SIN-1 (3-morpholino-sydnonimine), in the absence and presence of 5 and 10 mM of two new copper(I) complexes, copper(I)[4-(diphenylphosphane)benzoic-acid]- [hydrotris(4-bromo-1H-pyrazol-1-yl)borate]

complex and copper(I) 4-(diphenylphosphane)benzoic-acid]hydrotris(1,2,4-triazol-1-yl)borate synthesized by us. DNA damage was assessed using the comet assay — a rapid and sensitive, single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. The parameter tail moment, used as an index of DNA damage, showed that in all cases the two copper(I) complexes remarkably inhibited DNA strand breaks induced by the different nitrogen oxide species. These findings are consistent with the antioxidant character of copper(I) complexes and give additional information on the potential implications for their use as therapeutic agents.

Lecture

12 The role of proteins in propagation of radical-induced biological damage Janusz Gebicki Department of Biological Sciences, Macquarie University, Sydney, Australia

The identification of initial molecular targets of free radicals and other reactive oxygen species in living organisms is of considerable interest, because it would provide a rational approach to the inhibition of damage in biological systems at an early stage. There is considerable evidence that the first molecular targets in cells are proteins rather than DNA or lipids. Recent work

has demonstrated that proteins exposed to physiologically significant free radicals acquire semistable peroxide groups which can give rise to new radicals, inactivate enzymes, crosslink to DNA, and oxidise intra- and extracellular antioxidants. We have also shown that protein peroxides are generated in cultured cells incubated in the presence of hydroxyl or peroxyl radicals.

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39th Meeting of the Polish Biochemical Society

Our current studies are focused on reactions of the precursors of the peroxides, carbon-centred and peroxyl protein radicals. Pulse-radiolytic measurements carried out in collaboration with Dr. J. Gebicki’s Lodz group showed that these radicals are able to oxidise ABTS (2,2’-azinobis(3-ethyl-6-benzothiazoline sulpho4 9 nate)) with rate constants between 2x10 and 2.5x10 –1 –1 M s , depending largely on the molecular weight of the individual proteins. Direct measurement of the reaction of lysozyme radicals with ascorbate gave a rate 4 –1 –1 constant of 1.5x10 M s , demonstrating the possibility of rapid destruction of this important antioxidant in vivo. This was supported by results obtained by exposing mixtures of proteins and ascorbate or GSH to a

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flux of radiation-generated HO radicals, under conditions ensuring that the radicals reacted only with the proteins. This reaction generated protein carbon-centred and peroxyl radicals, which, in turn, caused the loss of ascorbate and GSH. Taken overall, these results suggest that cell damage by ionising radiation results in formation of reactive protein intermediates which constitute a new class of reactive oxygen species. References: Gebicki S, Gebicki JM (1993) Biochem J, 289: 743–749. Gebicki JM (1997) Redox Report, 3: 99–110. Gieseg S, Duggan S, Gebicki JM (2000) Biochem J, 350: 215–218.

Lecture

13 Antioxidant defence capacity in ageing — the case of centenarians Barbara K³apciñska

Katedra Nauk Fizjologiczno-Medycznych, Zak³ad Biochemii, Akademia Wychowania Fizycznego, ul. Mikolowska 72A, 40-065 Katowice

The free radical hypothesis of ageing is based on the assumption that endogeneous antioxidants defences of the organism are deficient and the residual prooxidants may cause oxidative damage to the cells even under normal physiological conditions. However, due to the complex and multifaceted character of the ageing process it is difficult to determine the relative contribution of individual causal factors to senescence.

The following aspects of ageing will be discussed: (1) age-associated trends in oxidant generation, antioxidant defences and repair of oxidative damage; (2) life-span potential and capacity for mitochondrial oxidant generation; (3) oxidative stress and glycemic regulation; (4) effects of dietary restriction, supplementation with antioxidants and physical activity on oxidative stress during ageing. Results of studies on antioxidant status in healthy centenarians will be presented.

Lecture

14 Lipid hydroperoxide cytotoxicity and detoxification 1

Witold Korytowski , Albert Girotti

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1 — Department of Biophysics, Faculty of Biotechnology, Jagiellonian University, ul. Gronostajowa 7, Kraków, Poland, 2 — Department of Biochemistry, Medical College of Wisconsin, Milwaukee, USA

Unsaturated membrane lipids in cells confronted with physical or chemical oxidative insults may undergo oxidative damage. Lipid hydroperoxides (LOOHs), including cholesterol (Ch)- and phospholipid (PL)-derived species are important non-radical intermediates in such processes. Once formed LOOHs can have a variety of fates which impact on the viability of a targeted cell. If LOOHs escape selenoperoxidase (GPX-4)-catalyzed detoxification (2-electron reduction), they may undergo iron-ion catalyzed 1-electron reduction to free radical species, thus triggering chain peroxidation reactions that can

exacerbate and broadcast initial oxidative insult. Alternatively, LOOHs — being more polar then parent lipids and longer lived then free-radical species — may translocate from site of origin to other membranes. An overall effect of such translocation will depend on the pro-/anti-oxidant status of the recipient membrane. We have investigated this aspect of LOOHs dynamics using photooxidized erythrocyte ghost membranes as LOOH donors, small liposomes as acceptors and HPLC with polarographic detection or HPTLC with phosphorimaging detection for analysis. Both ChOOHs and PLOOHs translocate much faster then

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parent (non-oxidized) lipids. The first-order rate constants for spontaneous transfer of individual ChOOH species increase with increasing polarity of peroxide. Toxicity of ChOOHs towards GPX-4-null COH-BR1 cells correlated with their transfer rate from liposomal donors. Similar observations had been

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made with various phospholipid peroxides. Transfer-acquired LOOHs sensitized membranes and cells to chain-peroxidation damage, supporting hypothesis that intra- and extracellular translocation can expand the cytotoxic range of LOOHs. Supported by KBN3P05A-5523 and by USPHS CA72630.

Lecture

15 NADPH oxidase and catalase-elements of plant response to infection Magdalena Krzymowska

Plant Biochemistry Department, Institute of Biochemistry and Biophysics, PAS, CEMB, ul. Pawiñskiego 5a, 02-106 Warsaw

Pathogen recognition by plants triggers multicomponent defense response. Early processes comprise changes of the ion permeability of the plasma membrane, protein phosphorylation/dephosphorylation and the production of reactive oxygen species (ROS) referred to as oxidative burst. ROS play a role in several aspects of the plant’s response to pathogen. They are involved in direct killing of pathogens. They reinforce the cell wall through lignification and oxidative cross-linking of its proteins. Eventually, they act as inter- and intracellular messengers in signal cascades leading to hypersensitive cell death, activation of defense-related genes, phytoalexin production, salicylic acid biosynthesis and establishment of systemic acquired resistance. A growing body of evidence suggests that generation of ROS upon pathogen attack is mediated by a membrane-bound

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NAD(P)H oxidase that resembles the phagocyte enzyme. As the first step the enzyme forms superoxide radicals which are then converted to oxygen and hydrogen peroxide either spontaneously or by an extracellular superoxide dismutase. Alternatively, the contribution of other enzymes to the oxidative burst, like peroxidase, amine oxidase or oxalate oxidase is postulated. A number of antioxidant enzymes such as superoxide dismutases, ascorbate peroxidases, peroxidases, glutathione reductases and catalases are involved in the specific detoxification of ROS. There are many reports that indicate that catalases may play a critical role in plant defense mechanisms. In general, the importance of catalase could reside both in its direct antioxidant activity and its ability to affect signal transduction pathways that entail H2O2 as a signal.

Lecture

Anti- and pro-oxidant aspects of thiols: a physico-chemical, biochemical and cellular point-of-view Pierre Leroy MAEM UMR, CNRS-UHP, Nancy, France

The protective action of thiols is associated with their high reducing potential (apparent E’° at pH 7.4 = –200 to –250 mV), that enables them to react with reactive oxygen species (ROS) either in a direct way or via enzymatic mechanisms. The pro-oxidative effects of thiols originate in the one-electron reduction of oxygen during their oxidation to disulfides, and in the formation of thiyl radicals. Thiol-dependent oxidative damage depends on the type of the thiol, the presence of transition metals (copper, iron), and on pH. Redox reaction of thiols occurs predominantly at the thiolate anion level; thus, at physiological pH, the nucleophilicity of a thiolate anion is inversely proportional to the pKa of the thiol (pKaSH).

The ability of cells to maintain high levels of reduced glutathione (GSH) is one of the main process to fight oxidative stress. However, this mechanism can induce resistance against anti-cancerous drugs in tumoral cells, and resistance of bacteria during chlorination. In the last decade, several works pointed out a pro-oxidant role of g-glutamyltransferase (GGT) linked to the metabolism of GSH in the presence of transition metals. This pro-oxidant effect is based on the autooxidation of cysteinylglycine, the GGT-generated metabolite of GSH, which produces thiyl and ROS. Studies performed with different cell models demonstrated that GGT-initiated production of ROS can result in oxidative damage of various lipid substrates, hu-

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39th Meeting of the Polish Biochemical Society

man LDL, microsomes, as well as oxidative mutagenesis and protein thiolation. By contrast, the knowledge about antioxidative and prooxidative effects of thiol drugs is poor. The family of thiol drugs contain mucolytic, chemoprotective, antiarthritic, antihypertensive and anticancer agents. The thiol moieties of such drugs may trigger antioxidative and prooxidative reaction, leading to beneficial or toxic effects.

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Phytochelatins (PCs) are a family of small cystein-rich peptides capable of binding heavy metals ions via their SH. Their general structure is [g-Glu-Cys]n-Gly (n = 2 to 8), they are synthesized from glutathione by g-glutamylcysteine dipeptidyl transpeptidase and they have been found in fungi, algae, in all plants species studied and in the nematode Caenorhabditis elegans. They are more and more extensively studied in relationship with cell response to metal stress.

Lecture

17 Gamma-glutamyltransferase: anti- or pro-oxidant enzyme? Pierre Leroy, Athanase Visvikis MAEM UMR, CNRS-UHP, Nancy, France

role of GGT is called “anti-oxidant” as it helps in maintaining the cellular GSH level and hence the intracellular redox balance. It is a major role of GGT as it has been demonstrated using cellular and animal models. We have recently demonstrated that under limiting cysteine concentrations GGT activity level is a determinant for the intracellular GSH level. On the contrary, extracellular GSH catabolism can also lead to pro-oxidant effects. The GGT pro-oxidant effect is based on the generation of CysGly, which contrary to GSH itself, is a redox labile metabolite. Autooxidation of CysGly in the presence of transition metals leads to pro-oxidant species such as thiyl and oxygen radicals. The GGT/GSH dependent oxidant stress modulates intracellular transduction pathways, it can induce critical transcription factors such as NF-kB, and it is thought to participate in oxidative mutagenesis, cell proliferation and tumor cell invasion and metastasis.

gamma-Glutamyltransferase (GGT) is a unique plasma membrane enzyme, which is able to initiate the degradation of extracellular glutathione (GSH) by cleavage of the gamma-glutamyl bond, allowing the supply of extracellular cysteine for intracellular synthesis of the tripeptide. GGT transfers the gamma-glutamyl moiety of GSH to an amino acid forming gamma-glutamyl-amino acid and cysteinylglycine. Cysteinylglycine is then degraded by dipeptidases into cysteine and glycine, both of which can be taken up by the cells and used for de novo GSH synthesis. In addition GGT participates in the GSH salvage pathway. In this pathway GGT transfers the gamma-glutamyl moiety of extracellular GSH to cystine forming gamma-glutamylcystine. gamma-Glutamylcystine is then directly taken up by the cells, and reduced to gamma-glutamyl cysteine and cysteine. gamma-Glutamyl cysteine can be directly used by glutathione synthetase thus bypassing the normally rate limiting step catalysed by GCL. This

Lecture

18 Nitric oxide regulates cytosolic labile iron pool by different mechanisms 1

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Pawe³ Lipiñski , Rafa³ Starzyñski , Jean-Claude Drapier , Cecile Bouton , Teresa Bart³omiejczyk , Barbara 3 1 4 4 Sochanowicz , Ewa Smuda , Agnieszka Gajkowska , Marcin Kruszewski 1 — Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzêbiec, 2 — Institut de Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette, France, 3 — Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Warszawa, 4 — Department of Experimental Hematology and Cord Blood Bank, Maria Sk³odowska-Curie Memorial Cancer Centre and Institute of Oncology, Warszawa

Mammalian cells, when exposed to the action of nitric oxide (NO), considerably alter their iron metabolism. Iron regulatory protein 1 (IRP1)-driven post-transcriptionally controlled regulation of ferritin (Ft) and transferrin receptor (TfR) synthesis is thought to be the best characterized pathway by which NO may alter cellular iron content and distribution. Strikingly, the

consequence of this regulation to the amount of iron in so called labile iron pool (LIP), a cytosolic pool of low molecular-weight iron complexes has never been studied in living cells. LIP constitutes a fraction of regulatory and metabolically active iron. On the other hand, LIP is widely implicated in generation of reactive oxygen species and oxidative modifications of bio-mo-

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Session 1. Biochemistry of oxidative stress

lecules. This dual biological role of LIP imposes a tight regulation of its level. The interplay between LIP and IRP1, and more precisely its iron-sulfur cluster is crucial for cell iron sensing. LIP level determines the [4Fe-4S] cluster status of IRP1 molecules and thus defines how IRP1 will function. In the absence of iron in LIP, binding of apo-IRP1 to iron regulatory elements (IREs) in the 5’UTR of Ft subunits mRNAs results in inhibition of translation whereas binding of IRP1 to IREs in the 3’ UTR of TfR mRNA protects this mRNA from degradation. Such a bi-directional but reverse regulation occurs in cells with high LIP level and is due to the lack of interaction between holo-IRP1 and IREs. By means of these opposite regulations cells rapidly restore their physiological LIP levels. Here, in order to investigate the effect of NO on the relationship between the modulation of IRP1 activities and LIP level, we used two closely related mouse lymphoma L5178Y (LY) sublines (LY-R and LY-S cells) that display two distinct patterns of both LIP and IRP1 levels. As a source of bioactive NO we used two NO-generating compounds from diazeniumdiolate family (called NONOates) displaying various, reproducible rates of NO release. We incubated LY cells with Sper/NO for 2 hours and with DETA/NO for 16 hours

2003

to mimic burst and extended exposure to NO, respectively. Under two exposure procedures, in both cell lines NO induced IRE binding activity of IRP1 and strongly inhibited its aconitase activity. This classical NO-mediated modulation of IRP1 activities was closely correlated with concomitant increase in LIP levels. We showed that the rise of LIP levels in LY cells treated with Sper/NO was not associated with any changes of the expression of neither Ft subunits nor the TfR molecule number on cellular membranes. We suggest therefore, that iron entering LIP during exposure of LY cells to Sper/NO, originates in a major part from NO-disrupted IRP1 [4Fe-4S] centres. In contrast, increase in LIP levels in DETA/NO-treated cells was very likely associated with down-regulation of Ft subunits expression, i.e. decreased degree of intracellular iron sequestration. Our experiments provide a new set of data demonstrating that NO can modulate IRP1 activities independently of fluctuations of LIP, a fraction of cellular iron thought to be a sensor for the IRP1/IRE regulatory mechanism. Furthermore, the finding of increase in LIP levels in mammalian cells exposed to NO opens a new clue to investigate biological mechanisms of NO-mediated cytotoxicity, e.g. possible links with oxidative stress.

Lecture

19

Three dimensional structure of actin-based membrane skeleton as revealed by electron microscopic computed tomography of rapidly-frozen, deep-etched plasma membrane 1

2

1

1

3

4

Nobuhiro Morone , Rinshi Kasai , Takahiro Fujiwara , Hiroshi Ike , Yukiko Hirata , Shigeki Yuasa , Yuji 5 6 5 1 Nishizawa , Takashi Wakabayashi , Jiro Usukura , Akihiro Kusumi 1 — Kusumi Membrane Organizer Project, Exploratory Research for Advanced Technology (ERATO), Science & Technology Corporation (JST), Nagoya, Japan, 2 — Department of Biological Science, Nagoya University, Nagoya, Japan, 3 — FEI Company Japan, Ltd., Tokyo, Japan, 4 — Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan, 5 — Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya University, Nagoya, Japan, 6 — Department of Cell Biology and Molecular Pathology, Medical University of Gdañsk, ul. Debinki 1, 80-211 Gdañsk

We have previously succeeded in visualizing the undercoat structure of the entire dorsal cell membrane over the whole cell, using rapid-freeze, deep-etch, immunoreplica electron microscopy. In this study, we further extended our research to obtain the high-resolution three-dimensional structure of the membrane undercoat by introducing computed tomography. For 3-D reconstruction, electron microscopic images of a sample tilted with respect to the horizontal plane were collected every 1.0° in the range of ± 70° (total 141 images) and used for 3-D rendering. The structure of the membrane skeleton mesh interacting with the plasma membrane was examined in the slices within 7–14 nm from the cytoplasmic surface of the membrane. Many cortical actin filaments were found to

be closely apposed to the plasma membrane, constituting the membrane skeleton, and compartmentalizing the plasma membrane. In cultured NRK cells, the com2 partment was 0.04 mm in area on average or 200 nm in the side length. Kusumi and Sako (1996) and Fujiwara et al. (2002) indicated that the plasma membrane is compartmentalized with respect to translational diffusion of proteins and lipids in the plasma membrane, due to the corralling effects of both the membrane skeleton and various transmembrane protein pickets anchored to the membrane skeleton. The median compartment size for diffusing lipids and proteins in NRK cells is 230 nm. Therefore, the compartment size determined by the diffusing molecules is in agreement with that directly determined by the EM tomography

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39th Meeting of the Polish Biochemical Society

method, supporting the membrane skeleton fence and anchored protein picket models. Such actin-based membrane skeleton was found to be linked to the rims of clathrin coats of the pits and the striated structures of caveolae. These results indicate that electron micro-

15

scopic computed tomography of rapidly-frozen, deep-etched, immunoreplicated plasma membrane is a new, useful method to study the plasma membrane undercoat structures.

Lecture

20

Iron metabolism and oxidative stress: Is hydrogen peroxide an intercellular signaling molecule? Sebastian Mueller Dept. Internal Medicine IV, University of Heidelberg, Bergheimer Str. 58, Heidelberg, Germany

Reactive oxygen species (ROS) not only damage proteins, lipids and carbohydrates, but are increasingly discussed in playing an important role in inter- and intracellular signaling. Therefore, their participation in disease may also be related to the disruption/modulation of redox-sensitive signaling pathways. Many experimental difficulties arise from limitations in the detection of unstable ROS in cells and tissues and the lack of appropriate models. We have previously established an ultra-sensitive chemiluminescence technique for the detection of H2O2 that is considered a major candidate in signaling. By taking advantage of this method, compartmentation data have been obtained and an enzymatic system (GOX/CAT) has been developed to generate and maintain steady-state H2O2 concentrations for up to two days in cultured cells, thus mimicking in vivo inflammatory conditions. Furthermore, we successfully applied the GOX/CAT system to study the signaling role of H2O2 on iron metabolism. It is shown in vitro and in vivo that H2O2 mediates the uptake of

transferrin bound iron via the transferrin receptor (TfR). The underlying mechanism is mostly due to an activation of the central iron masterswitch IRP-1 (iron regulatory protein 1) that stabilizes TfR mRNA by binding to its iron responsive elements (IRE’s). Taking together our studies on H2O2 compartmentation, metabolism and iron signaling, H2O2 should presently viewed as an intercellular messenger molecule playing a special role in the communication between inflammatory and parenchymal cells. The presentation will briefly discuss present strategies of our lab to better understand H2O2 mediated signalling that include the generation of a transgenic mouse model expressing the H2O2-producing enzyme glucose oxidase (GOX) from the fungus Aspergillus niger in a liver-specific and tetracycline-dependent fashion and the recent development of a murine and human microarray (“liver chip”) to study ROS-mediated gene expression pattern in cell culture and animal models.

Lecture

21 Aldehyde reactivity with aminoacid side-groups in peptide structures Jerzy Naskalski

Katedra Biochemii Klinicznej, Collegium Medicum, Uniwersytet Jagielloñski w Krakowie, ul. Kopernika 15B, 31-501 Kraków

Tryptophane (a-amino, b-indoloacetic acid) is one of the most reactive amino acids. At pH 4.0–6.0 tryptophane amino group reacts with aldehydes producing Schiff-bases. On the other hand if the a-amino group is blocked by acetylation, tryptophane reacts with aldehydes yielding carboline derivatives (carboline 1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid). Finally, in the presence of aldehydes, tryptophane reacts yielding dimeric, trimeric or polymeric condensation products. Our studies were focused on tryptophane indoil residue activity in water solutions and pH being in the

range of physiological fluids. Reactions of tryptophane derivatives with different substitutes of the side chain in position 3, as it is in N-acetyltryptophane, were investigated. If the position at nitrogen 1 was blocked by the substitutes, no reaction with aldehydes was observed. Thus, most probably nitrogen at position 1 is preferable as an attack target in reactions with aldehydes. Indoylo residue reacts with aldehydes with various molecular structures. The obtained product was tryptophane dimer. Additional modifications consist of hydroxyalkyl or hydroxyaryl derivatives of indoyl residues.

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Session 1. Biochemistry of oxidative stress

Tryptophane indoil residue reaction with aldehydes occurs in several stages: at first an unstable hydroxyalkyl derivative, presumably being a substitute of aldehyde to pyrrole ring nitrogen is produced. This compound, however, rapidly reacts further, producing water soluble dimer molecule. If the reaction mixture was left over for several days in room temperature, a water insoluble reaction products are formed. These are most probably, the tryptophane polymerization re-

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action products. As the studied reactions were carried out in physiologic pH and diluted saline-phosphate buffer solution, it is conceivable that tryptophane polymerization may occur in living systems as well. Particularily in cells producing excessive amounts of aldehydes, and condensation of tryptophane residues may also contribute to oxidative changes in protein structures.

Lecture

22

NADH influences redox state of hemoproteins with an involvement of reactive oxygen species 1

1

1

2

2

Robert Olek , Jêdrzej Antosiewicz , Jerzy Popinigis , Gian Carlo Caulini , Donatella Fedeli , Giancarlo 2 Falcioni 1 — Department of Bioenergetics, Jedrzej Sniadecki University School of Physical Education and Sport, ul. Wiejska 1, 80-336 Gdañsk, 2 — Department of MCA Biology, University of Camerino, Camerino, Italy

Hemoglobin and myoglobin are respiratory heme-proteins able, when they are in the ferrous forms, to undergo a reversible reaction with molecular oxygen. However, oxyhemoglobin and oxymyoglobin can turn to ferric form (Hb3+ and Mb3+) which are unable to bind oxygen. This conversion is associated with superoxide anion production and thereby with products such as hydrogen peroxide and hydroxyl radicals, which can derive from superoxide anion itself. It is known that reduced pyridine nucleotides are capable of enzymatic reduction of met-hemoglobin; non-enzymatic reduction of met-Hb requires the presence of an electron carrier. In this study, we have investigated the influence of NADH on the redox state of both HbA and

Mb and the role of superoxide dismutase (SOD) and catalase (CAT) in this process. Our data show that NADH can increase the autoxidation rate of HbA and Mb in a pH-dependent fashion. During this process, this cofactor is itself oxidized. The presence of SOD and /or CAT can inhibit this result. Our data indicate that the reduced pyridine nucleotide may influence the redox state of both the hemoproteins by a mechanism that probably involves free radical species. This result, referred to myoglobin, could have a physiological importance, given that in the muscle the ratio between NADH and hemoproteins is higher with respect to erythrocyte.

Lecture

23

Oxidative DNA damage in cancer patients; a cause or a consequence of the disease development? Ryszard Oliñski, Daniel Gackowski, Rafa³ Ró¿alski, Marek Foksiñski, Karol Bia³kowski Katedra i Zak³ad Biochemii Klinicznej, Akademia Medyczna, ul. Kar³owicza 24, 85-092 Bydgoszcz

A wide variety of oxidative DNA lesions are present in living cells. One of the best known lesions of this type is 8-oxoguanine (8-oxoGua) which has been shown to have mutagenic properties. Our recently published results concerning an influence of antioxidative vitamins and labile iron pool on the background level of 8-oxoGua in cellular DNA [1–4] are discussed and oxidative damage to free nucleotide pool as a possible source of 8-oxo-2’-deoxyguanosine in DNA and urine is described. An involvement of 8-oxoGua in the origin and/or progression of cancer is reviewed. It is con-

cluded that a severe oxidative stress manifested as a high level of 8-oxoGua in cellular DNA as well as in urine of cancer patients is a consequence of development of many types of cancer. Although at present it is impossible to answer directly the question concerning involvement of oxidative DNA damage in cancer etiology it is likely that oxidative DNA base modifications may serve as a source of mutations that initiate carcinogenesis (i.e. they may be causal factors responsible for the process).

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39th Meeting of the Polish Biochemical Society

References: 1. Gackowski D, Kruszewski M, Bartlomiejczyk T, Jawien A, Ciecierski M, Olinski R (2002) J Biol Inorg Chem, 7: 548–550. 2. Oliñski R, Gackowski D, Foksiñski M, Ró¿alski R, Roszkowski K, Jaruga P (2002) Free Rad Biol Med, 33: 192–200.

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3. Rozalski R, Gackowski D, Roszkowski K, Foksinski M, Olinski R (2002) Cancer Epidemiology, Biomarkers & Prevention, 11: 1072–5. 4. Gackowski D, Banaszkiewicz Z, Rozalski R, Jawien A, Olinski R (2002) Intern J Cancer, 101: 395–397.

Lecture

24

Melatonin as an antioxidant: Biochemical mechanisms and pathophysiological implications Russel Reiter Department of Cellular and Structural Biology, University of Texas Health Science Center, ul. 7703 Floyd Curl Drive, San Antonio, USA

N-Acetyl-5-methoxytryptamine (melatonin) is a molecule that is endogenous, produced in all organisms and is also consumed in the diet. It has a variety of functions including circadian rhythm regulation and, furthermore, it has newly-discovered, potent and ubiquitous antioxidative actions. Melatonin directly detoxifies a variety of oxygen and nitrogen-based reactants including the devastatingly toxic hydroxyl radical. The mechanisms whereby melatonin scavenges radicals have been in part defined, as have been the resulting products. Some of the major metabolites that are formed when melatonin incapacitates toxic reactants include cyclic 3-hydroxymelatonin and N1-acetyl-N2-formyl-5-methoxykynuramine. Interestingly, these products also function as radical scavengers. Thus, not only is the parent antioxidant, i.e., melatonin, an effective free radical scavenger, but the products that are formed are as well. This antioxidant cascade greatly expands the number of free radicals a single melatonin molecule can scavenge. Beyond this, melatonin stimulates the synthesis of another impor-

tant intracellular antioxidant, glutathione, as well as improving the activities of several antioxidative enzymes. Finally, melatonin probably reduces electron leakage from the mitochondrial electron transport chain thereby lowering free radical generation and elevating ATP production. Besides defining the mechanisms of melatonin as a direct free radical scavenger and indirect antioxidant, many groups have examined its ability to reduce oxidative stress under a wide variety of experimental conditions. Melatonin is readily absorbed when administered via any route and it crosses all morphophysiological barriers with ease, e.g., the placenta and the blood-brain barrier; furthermore, melatonin has no known toxicity. Examples of experimental free radical-mediated conditions where melatonin has been found to be highly beneficial include models of Alzheimer disease, Parkinsonism and ischemia/reperfusion injury. In each of these situations both physiologically-relevant and pharmacological doses of melatonin have been shown to significantly reduced the associated oxidative damage.

Lecture

25

Interaction of plasmenylcholine with singlet oxygen and free radicals — a model system for determination of plasmalogens susceptibility to oxidation 1

1

1

1

2

Tadeusz Sarna , Radoslaw Klosinski , Anna Pawlak , Marta Wrona , Junhwa Shin , David Thompson

2

1 — Wydzial Biotechnologii, Zak³ad Biofizyki, Uniwersytet Jagiellonski, ul. Gronostajowa 7, 30-387 Kraków, 2 — Department of Chemistry, Purdue University, West Lafayette, USA

A growing body of experimental evidence supports the hypothesis that the vinyl ether bond, present in plasmalogens, may protect polyunsaturated fatty acids (PUFA) from oxidative damage by serving as a sacrificial trap for reactive oxygen species. Such a protection may be of particular importance in cardiac tissues and

in the central nervous system that contain high concentrations of PUFA and plasmalogens. In this study, we investigated the interaction of semi-synthetic plasmenylcholine (PlsC) with singlet oxygen, generated by photosensitized energy transfer, using selected photosensitizing dyes in homogenous solu-

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Session 1. Biochemistry of oxidative stress

tions of PlsC and, for comparison, of cholesterol or other peroxidizable and non-peroxidizable lipids, and in heterogenous liposomal sytems made of PlsC, DMPC and/or cholesterol. Progress of lipid peroxidation was monitored by oxygen EPR-oximetry, iodometric determination of total lipid hydroperoxides and by HPLC-EC(Hg) detection of characteristic cholesterol hydroperoxides — specific products of cholesterol interaction with singlet oxygen or free radicals. Rate constants of the interaction of singlet oxygen with PlsC and other lipids were determined by measurement of singlet oxygen lifetime in the absence and presence of the lipids using time-resolved phosphorescence at 1270 nm. To determine the efficiency of PlsC to interact with oxidizing radicals, pulse radiolysis measurements of

2003

the radical lifetime in the absence and presence of Triton X-100 solubilized plasmenylcholine (and other lipids), were carried out. Our data indicate that singlet oxygen interacts with PlsC 1.5–2 orders of magnitude faster than with cholesterol or methyl esters of different PUFA. Although we were unable to detect any measurable amounts of PlsC hydroperoxides, when this phospholipid was exposed to typical free radical initiators, results of the pulse 2– radiolysis measurements clearly showed Br and CClO3 interacted with PlsC about 30–300 times more efficiently than with linolenic acid methyl ester. Our quantitative study provides an unambigous evidence for high reactivity of plasmenylcholine with singlet oxygen and certain oxidizing radicals.

Lecture

26 Is green tea a universal antioxidant? El¿bieta Skrzydlewska, Justyna Ostrowska Zak³ad Chemii Nieorganicznej i Analitycznej, Akademia Medyczna, ul. Mickiewicza 2, 15-230 Bia³ystok

An imbalance in the prooxidant/antioxidant status of a cell with an excess of the former potentially leads to the formation of oxidative stress, which appears to cause or participate in the pathogenesis of several human diseases including neurodegenerative disease, cancer, aging, cardiovascular disease and inflammatory disease. Intervention with antioxidants to reduce oxidative stress may potentially prevent these diseases or slow their pathogenesis. Tea is one of the most popular beverages consumed worldwide. Tea leaves are primarily manufactured as green, black or oolong. Green tea is the nonoxidized/nonfermented product and contains several polyphenolic components, mainly flavonoids such as epicatechin, epicatechin gallate, epigallocatechin and epigallocatechin gallate. Some epidemiological studies have found an inverse association between green tea consumption and the risk of coronary disease, stroke, cancer, and Parkinson and Alzheimer disease. Among the principal properties that may account for the potential health benefits of green tea is

its antioxidant activity. Several in vitro studies have demonstrated that green tea flavonoids can scavenge superoxide, hydroxyl and peroxyl radicals and inhibit lipid peroxidation in different system, i.e., phospholipid membranes, microsomes and mitochondria, human erythrocytes, low-density lipoprotein and human plasma depleted of ascorbate. Several mechanisms may account for the antioxidant activity of green tea flavonoids in addition to free radical scavenging, viz. chelating of transition metal ions, inhibition of oxidant enzymes or production of free radicals by cells, and regeneration of a-tocopherol from the a-tocopheroxyl radical. Therefore the consumption of tea has been proposed as a useful practice to limit oxidative damage in the body. However, studies of tea consumption in animals and humans have produced conflicting results, suggesting that the evidence for antioxidant activity of tea flavonoids in vitro cannot be extrapolated to the in vivo situation.

Lecture

27 Functional consequences of oxidative membrane damage Guenther Stark, Frank Killig Biochemistry, University of Konstanz, Konstanz, Germany

The interaction of reactive oxygen species with biological membranes is known to produce a great variety of different functional modifications. Part of these

modifications may be classified as direct effects. They are due to direct interaction of the reactive species with the molecular machinery under study with a subse-

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39th Meeting of the Polish Biochemical Society

quent chemical and functional modification of these molecules. An important part of the observed functional modifications are, however, indirect effects. They are the consequence of an oxidative modification of the environment of biological macromolecules. Lipid peroxidation — via its generation of chemically reactive products — contributes to the loss of cellular functions through the inactivation of membrane enzymes and even of cytoplasmic (i.e. water soluble) proteins. Oxidation of membrane lipids may, however, also increase the efficiency of membrane functions. This was observed for a series of transport systems. Lipid peroxidation was accompanied by activation of certain types of ion channels and of ion carriers. The effect is due to an increase of the polarity of the membrane interior by accumulation of polar oxidation products. The concomitant change of the dielectric constant, which

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may be detected via the increase of the membrane capacitance, facilitates the opening of membrane channels and lowers the inner membrane barrier for the movement of ions across the membrane. The conclusions were derived from experiments performed with different systems: a) model systems (gramicidin A, alamethicin, amphotericin B, valinomycin) in planar lipid membranes, b) native ion channels either reconstituted in lipid membranes or investigated in their natural environment by the patch-clamp method, c) two important ion pumps, the Na/K-ATPase and the sarcoplasmic reticulum. Reactive oxygen species were generated by absorption of ionizing radiation (water radiolysis) or by visible light in the presence of membrane-active photosensitizers.

Lecture

28 Nitric oxide and poly(ADP-ribose) polymerase in signal transduction and neurodegeneration 1

1

1

1

Joanna Strosznajder , Henryk Jêœko , Ma³gorzata Chalimoniuk , Agata Zambrzycka , Robert Strosznajder

2

1 — Zak³ad Komórkowej Transdukcji Sygna³u, Instytut Medycyny Doœwiadczalnej i Klinicznej, PAN, ul. Pawiñskiego 5, 02-106 Warszawa, 2 — Zak³ad Neurofizjologii, Instytut Medycyny Doœwiadczalnej i Klinicznej, PAN, ul. Pawiñskiego 5, 02-106 Warszawa

Nitric Oxide (NO) is a very potent inter- and intracellular messenger. NO is involved in neurotransmitters release and in transduction of information from receptors into the nucleus including the new nuclear target poly(ADP-ribose) polymerase (PARP-1, EC 2.4.2.30). This nuclear enzyme is activated in response to DNA breaks and it regulates DNA repair and gene expression. However, its excessive activation leads to NAD and ATP depletion and cell death. Our last studies concentrated on gene expression and activity of different NO sythases (NOS) isoforms and PARP during aging and ischemia and on their role in cell death. The data indicated aged related enhancement of NO release by constitutive isoforms of NOS, with higher gene expression for eNOS and lower level

of mRNA for nNOS comparing to adult. It seems that transient induction of iNOS during aging may be responsible for the downregulation of the gene for nNOS. The higher release of NO and the other free radicals during brain aging and ischemia-reperfusion injury disturbs DNA integrity and enhances PARP activity that is further modulated differently depending on the age, brain region, the type and time of ischemia. The significant enhancement of PARP activity during brain ischemia-reperfusion may be responsible for the alteration of transcriptional factors NF-kB, p53, energy metabolism, release of apoptotic inducible factor (AIF) from mitochondria and apoptotic and necrotic cells death.

Lecture

29 DNA polymerase a is essential for genomic stability Motoshi Suzuki

Division of Molecular Carcinogenesis Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, 466-8550 Nagoya, Japan

We have examined the biological impact of mutating a conserved Leu residue (L868) in the palm region of S. cerevisiae DNA polymerase a. An L868F mutant of

S. cerevisiae DNA polymerase a efficiently bypasses a cis-syn cyclobutane pyrimidine dimer (CPD), extending the 3’-T 28,000-fold more efficiently than wild type. The

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Session 1. Biochemistry of oxidative stress

L868F polymerase a mutant has an error frequency of 3 in 100 nucleotides and a replication accuracy 570-fold lower than wild type. In vivo, this mutant did not confer sensitivity to UV or methyl methanesulfonate, but confers a mutator phenotype and is associated with increased genomic instability. Transitions are the predominant mutation in the in vitro mutation spectrum of L868F polymerase a, but transversions are the most common mutation in vivo at the trp1 locus. In translesion DNA polymerases, the amino acid equiva-

2003

lent to Leu868 is F34, suggesting that the intrinsic low fidelity of translesion polymerases might be related to the presence of phenylalanine at this position. The data suggest that F34 and L868 palm residues in translesion and replicative DNA polymerases, respectively, may have played a role in the functional evolution of these enzyme classes. In addition, this study suggests that high fidelity DNA synthesis by pol a is essential for maintaining genomic stability.

Lecture

30 Mitochondrial genome single nucleotide polymorphisms and their phenotypes Masashi Tanaka Department of Gene Therapy, Gifu International Institute of Biotechnology, Kagamigahara, Gifu, Japan

Polymorphisms in the human mitochondrial genome have been used for elucidation of the phylogenetic relationships among various ethnic groups. Because analysis by mitochondrial genetics has detected pathogenic mutations causing mitochondrial encephalomyopathy or cardiomyopathy, most of the mitochondrial single nucleotide polymorphisms (mtSNPs) found in control subjects have been regarded as merely normal variants. We cannot exclude, however, the possibility that the mitochondrial functional differences among individuals are ascribable at least in part to the mtSNPs of each individual. Human life span in ancient history was much shorter than that at the present time. Therefore it is reasonable to speculate that certain mtSNPs that predispose one toward susceptibility to adult- or elderly-onset diseases, such as Parkinson’s disease and Alzheimer’s disease, have never been a target for natural selection in the

past. Similarly, thrifty mtSNPs that had been advantageous for survival under severe famine or cold climate conditions might turn out to be related to satiation-related diseases, such as diabetes mellitus and obesity. To examine these hypotheses, we have constructed the mtSNP database (http://www. giib.or.jp/ mtsnp/index_e.html) by sequencing the entire mitochondrial genomes from 672 subjects: 96 each of 7 groups, i.e., centenarians, young obese or non-obese subjects, diabetic patients with or without major vascular involvements, patients with Parkinson’s disease, and those with Alzheimer’s disease. We have identified mtSNPs associated with age-related conditions such as longevity, Parkinson’s disease, and Alzheimer’s disease, as well as those related to energy metabolism such as obesity, thinness, and type-2 diabetes, or to atherosclerosis.

Lecture

31

Proteomic, transcriptomic, signal transduction and functional studies of stressinduced premature senescence (SIPS) & potential role of sips in human aging Olivier Toussaint Unit of Cellular Biochemistry & Biology, University of Namur (FUNDP), Rue de Bruxelles, 61, Namur, Belgium

SIPS occurs in several proliferative cell types after subcytotoxic exposure to tert-butylhydroperoxide (t-BHP), H2O2, ethanol, UV-B, etc. TGF-b1 overexpression triggers the induction of several biomarkers of replicative senescence (RS) in human diploid fibroblasts (HDFs) within 72 hr after H2O2 or UV-B stress. H2O2-induced p38MAPK phospho-

rylation triggers a sustained overexpression of TGF-b1 through a sustained activation of ATF-2 transcription factor, due to establishment of a regulatory loop between TGF-b1 overexpression and sustained p38MAPK phosphorylation. At 24 hr after stress, ATF-2 interacts with hypophosphorylated retinoblastoma protein, which allows the biomarkers of RS to

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39th Meeting of the Polish Biochemical Society

appear (Free Rad Biol Med, 2000 & 2002; J Cell Sci, 2000; J Biol Chem, 2001; Int J Biochem Cell Biol, 2002). H2O2- and UV-B-induced SIPS also takes place in HDFs with telomerase activity. Very limited mean telomere shortening is observed in these conditions (FEBS Lett, 2002a; FEBS Lett, 2001). High resolution 2-D gels and mass spectrometry allowed to identify 30 proteins differentially expressed in SIPS induced by ethanol and t-BHP and/or in RS (FEBS Lett, 2002b;

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Biochem Pharmacol, 2002). Lastly we started transcriptomic studies with dedicated low density cDNA arrays for characterising the establishment of SIPS, and we started functional studies for investigating the role of the genes that are differentially expressed in SIPS and/or RS. These studies help the understanding of the mechanisms of appearance of SIPS and allow to hypothesise a potential role of SIPS in in vivo aging.

Lecture

32

Structural analysis of the membrane undercoat in cultured neural growth cone 1

2

3

1

3

Jiro Usukura , Yoji Kawano , Nobuhiro Morone , Yuji Nishizawa , Akihiro Kusumi , Kozo Kaibuchi

2

1 — Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya University, Nagoya, Japan, 2 — Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan, 3 — Kusumi Membrane Organizer Project, Exploratory Research for Advanced Technology (ERATO), Science & Technology Corporation (JST), Nagoya, Japan

Freeze-etching immuno-replica method was available for three-dimensional analysis of membrane undercoat of neural growth cone. Membrane undercoat consisted of actin filaments, microtubules, actin related proteins (Arp2/3) and small amount of spectrin. Those filaments formed complicated meshwork and attached closely onto the cytoplasmic surface of the plasma membrane. It is very interested what and how neuronal growth factors are associated in this membrane undercoat. We investigated immunocytochemically localization of axon guidance molecules, Sra-1, WAVE-1, and CRMP-2. In particular, we focused on CRMP-2 (Collapsing Response Medi-

ator Protein-2) because it was involved in initial formation of axon. All these axon guidance molecules were localized at actin filaments rich regions as well as the membrane undercoat. CRMP-2 was predominantly in membrane undercoat and stress fibers of growth cones and growing processes. Obviously, immuno-labeling was found on the actin fiber at EM level. Although the exact function of CRMP-2 has not been unknown, they control growing axon by regulating acin filaments assembly together with Sra-1 and WAVE-1, taking it into account that CRMP-2 is substrate of Rho-kinase.

Lecture

33 Control mechanisms on the size and distribution of mitochondria in the cell Takashi Wakabayashi Department of Cell Biology and Molecular Pathology, Medical University of Gdañsk, ul. Dêbinki 1, 80-211 Gdañsk

In the present study I have tried to overview the mechanisms which control the size and distribution of mitochondria in the cell. Data have been accumulated to demonstrate that spatial distribution and sizes of mitochondria in the cell are under the control of various genes detected mainly in yeast cells. Some of these genes have been shown to exert their actions via the connection with certain components of cytoskeleton. 1) Genes controlling the size and distribution of mitochondria in the cell. Gene products detected in mam-

malian cells, related to the size and spatial distribution of mitochondria are Drp1p, KIF1B and KIF5B 2) Cytoskeletons and mitochondrial distribution in the cell. A body of data is available in the literature demonstrating colocalization of mitochondria with cytoskeletons besides those in connection with genes. 3) Structural changes of mitochondria related to apoptosis. Structural changes of mitochondria related to apoptosis include: swelling, the megamitochondria formation and perinuclear clustering.

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Session 1. Biochemistry of oxidative stress

2003 Lecture

34 The switch mechanism of the cell death mode from apoptosis to necrosis Takashi Wakabayashi Department of Cell Biology and Molecular Pathology, Medical University of Gdañsk, ul. Dêbinki 1, 80-211 Gdañsk

To solve the mechanism of the transition of cell death mode from apoptosis to necrosis is one of the most important subjects in the research field of apoptosis. Numerous data are available in the literature describing that some chemicals at low concentrations induce apoptosis while they induce necrosis at higher concentrations. In extreme cases, apoptotic and necrotic changes have been shown to co-exist in one single cell. However, careful examination of data published in the literature has revealed that little attention has been paid to “secondary necrosis” which occurs to culture cells after they detach from the culture dish. In the present study we have examined time-dependent changes in the cell death mode from apoptosis to necrosis using menadione (MEN), an anti-cancerous drug, and osteosarcoma 143B cells excluding a possible involvement of “secondary necrosis”. The population of apoptotic cells judged by FITC-annexin V and propidium iodide (PI) double

staining reached its maximum at 6 hr after 100 mM MEN treatment followed by an abrupt decrease at 8 hr, while that of necrotic cells continuously increased reaching 90% at 24 hr. Electron microscopically, cell attached to the culture dish at 6 hr after MEN treatment consisted of two different types of cells: cells with typical apoptotic features and those with condensed nuclei and swollen cytoplasm designated as “intermediate cells”. Cell attached to the culture dishes at 8 hr after the treatment consisted exclusively of “intermediate cells”. Distinct decreases in intracellular levels of ATP and caspase-3-like activities and remarkable elevation of intracellular levels of superoxide, which was partly suppressed by NAD(P)H oxidase inhibitors, occurred between 6 and 8 hr after MEN treatment. The present study indicates that time-sequential studies on the cell death mode are essential and also suggests a possible involvement of NAD(P)H oxidase in the switch mechanism.

Lecture

35

Nitric oxide and cytoskeleton: transformation of gaseous signal into structural rearrangements in plant cells 1

2

Przemys³aw Wojtaszek , Dieter Volkmann , Frantisek Baluska

2

1 — Department of Biochemistry, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, ul. Fredry 10, 61-701 Poznañ, 2 — Department of Plant Cell Biology, Institute of Botany, Rheinische Friedrich-Wilhelms University Bonn, Kirschallee 1, 53115 Bonn, Germany

Nitric oxide (NO) is labile gaseous signaling molecule which has been implicated in the regulation of many physiological processes, both in animals and plants [1]. The best studied NO function in plants is the involvement in signalling and activation of defence responses of plants subjected to pathogen infection. Interestingly, in most cases activity of NO is tightly coupled to the action of reactive oxygen species, in particular hydrogen peroxide. Although the role of NO in regulation of plant growth and development has been proposed quite a long time ago, it is only in recent years that a growing body of supporting evidence is becoming to accumulate. It is now apparent that NO is participating in such distant processes as regulation of stomatal opening and closure, seed germination, delaying of senescence, cell elongation, and others. In most cases it is assumed that in plants, similarly like in animal cells, activation of guanylate cyclase by NO results in the generation of cGMP which then stimulates the biosynthesis of cADPR. It is suggested, however,

that in some cases NO can bind to other proteins and exert its physiological effects more directly. One line of evidence comes from the analysis of NO generation stimulated by mechanical stimuli, both in animal and plant cells. The other data indicate that in animal cells NO regulates actin reorganization [2] and, in this way, affects signalling pathways, e.g. activities of MAPK kinases [3]. In this lecture, preliminary data will be presented demonstrating that similar phenomena can occur in plant cells. We will show cell-type specific reorganization of actin cytoskeleton in maize root tips subjected to the action of various NO-releasing substances and other chemicals related to NO-signalling pathways. References: 1. Wojtaszek P (2000) Phytochemistry, 54: 1–4. 2. Ke XC et al. (2001) Biochim Biophys Acta, 1539: 101–113. 3. Ingram AJ et al. (2000) J Biol Chem, 275: 40301–40306. The research presented was supported by Alexander von Humboldt Fellowship to PW.

2003

39th Meeting of the Polish Biochemical Society

23 Lecture

36 Oxidative stress involvement in proapoptotic signalling of 4-octanoyl-TEMPO Micha³ WoŸniak Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk

Results of recent years underscore some antioxidants as potent inducers of apoptosis. The aim of present study was to define relationship between oxidative stress dependent proapoptotic signaling of acyl derivatives of piperidine aminoxyl and their chemical structure differing in length of acyl chain at 4th position of piperidine ring of 4-OH-TEMPO. Human osteosarcoma 143B cells were incubated with 4-OH-TEMPO, 4-octanoyl-TEMPO and 4-palmityl-TEMPO respectively. Surprisingly out of all chemical tested only 4-octanoyl-TEMPO exhibited cytotoxic properties in tested cells causing 50% of apoptotic cell death within 24 h of incubation. 4-Octanoyl-TEMPO has been found to induced reactive oxygen species (ROS). Inhibitor of NADPH oxidase

— diphenylene iodonium (DPI) attenuated release of ROS, supporting participation of this enzyme in oxidative stress. We observed loss of asymmetry of plasma membrane phospholipids — detected by Annexin V FITC staining and increase of caspase-3 activity. On the other hand 4-palmitoyl-TEMPO did not show any of these cytotoxic activities and displayed strongly immobilized EPR spectra in cell plasma membrane. EPR studies on lipid rafts model revealed immobilization of palmitoyl derivative of 4-OHTEMPO and surprisingly high mobility of octanoyl derivative. Collectively, the above results support high biological impact of chemical structure of aminoxyl on proapoptotic signaling of molecule starting from its interaction with cell membrane components.

Lecture

37 Membrane effects of oxidative damage of mammalian cells 1

1

2

Ilya Zavodnik , Elena Lapshina , Leu Zavodnik , Maria Bryszewska

3

1 — Laboratory of Membrane Biochemistry, Institute of Biochemistry, National Academy of Sciences of Belarus, Grodno, Belarus, 2 — Laboratory of Experimental Hepatology, Institute of Biochemistry, National Academy of Sciences of Belarus, Grodno, Belarus, 3 — Department of General Biophysics, University of Lodz, £ódŸ

The numerous effects of reactive oxygen, nitrogen and chlorine species in mammalian cells ranging from rapid cell mortality to regulation of cell signal pathways are well known. At the same time the chemistry and biochemistry of oxidative processes in the living cell as well as the molecular targets for oxidative modifications are not completely understood. The aim of this study was to investigate the role of membrane damage in cell impairments under oxidative stress. Using the different types of mammalian cells we examined the features of interactions of reactive oxygen (tert-butyl hydroperoxide, tBHP), nitrogen (sodium nitrite) and chlorine (hypochlorous acid, HOCl) species with cellular components. Well known model oxidant tBHP oxidising red blood cells (RBC) changed the cell glutathione pool, induced membrane lipid peroxidation and significant membrane hyperpolarisation due to selective potassium leakage and cell morphological transformations (vesiculation, exinocytosis). The inhibition of proliferation (reproducible cell death) of Chinese hamster B14 fibroblasts under tBHP treatment may be explained by tBHP genotoxicity (DNA damage as revealed by Comet assay), cell redox equilibrium distur-

bance and membrane structural modifications resulting in ion-nonselective pore formations. In vitro human erythrocyte exposure to HOCl (100–1000 mM), an extremely toxic biological oxidant, led to rapid oxidation of reduced glutathione and the inhibition of the membrane Na-, K-, Mg-ATP-ases resulting from direct oxidation of essential residues of enzyme (thiol groups) and structural rearrangements of the membrane. The HOCl-induced colloid-osmotic haemolysis preceded by rapid cell morphological transformations was apparently mediated by transient pore formation and altered membrane electrolyte permeability. The estimated pore radius was approximately 0.7 nm and the average number per cell was 0.01. Hyperpolarisation of erythrocyte membranes and an increase in membrane rigidity have been shown as a result of RBC oxidation by sodium nitrite. The photochemical oxidation of band 3 protein plays a significant role in the formation of haemolytic holes and in the photosensitized haemolysis. We can conclude that different oxidative agents produce oxidation of intracellular components, complex structural modifications of cellular membrane and cell morphological transformations. Disturbance

24

Session 1. Biochemistry of oxidative stress

in passive membrane permeability due to pore formation as well as inhibition of active ion pumps may be one of the dramatic cell oxidative impairments. The ef-

2003

fect of oxidant depended on the primary target in the cell and the sequence of cellular and membrane events.

Oral Presentation

38

Mechanisms of regulation of vascular endothelial growth factor synthesis by hydrogen peroxide and overexpression of copper-zinc superoxide dismutase: role of heme oxygenase-1 1

2

3

3

4

Jaros³aw Cisowski , Jolanta Grzenkowicz-Wydra , S. Cheng , Anupam Agarwal , Alicja Józkowicz , Anna 2 1 Podhajska , Józef Dulak 1 — Department of Cell Biochemistry, Faculty of Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, 2 — Intercollegiate Faculty of Biotechnology, University of Gdañsk and Medical Academy, Gdañsk, 3 — University of Florida, University of Florida, Gainesville, USA, 4 — Department of Molecular Genetics and Genetic Engineering, Jagiellonian University, ul. Granostajowa 7, 30-387 Kraków

Background: Cu,ZnSOD (SOD1) is an enzyme involved in dismutation of superoxide radical to hydrogen peroxide (H2O2). Heme oxygenase-1 (HO-1), which degrades heme to carbon monoxide (CO), iron and biliverdin, is a H2O2-inducible enzyme. Recently we showed that HO-1 overexpression augmented VEGF generation and that CO is responsible for this effect. Here we determined the involvement of HO-1 in H2O2-induced synthesis of VEGF. Methods and Results: VEGF production in rat vascular smooth muscle cells and murine NIH 3T3 fibroblasts was dose-dependently potentiated by exogenous H2O2 (50–400 mM). This effect was abolished by exogenously added catalase (1 U/ml). Both H2O2 and overexpression of SOD1 using plasmid vector led to VEGF promoter activation, as determined by increased synthesis of VEGF promoter-driven reporter gene. VEGF protein synthesis was significantly enhanced in 3T3 cells stably transfected with SOD1. Expression of HO-1 paralleled the induction of VEGF after treatment

with H2O2 and HO-1 expression was enhanced in SOD1-overexpressing cells. Hemin, an inducer of HO-1 enhanced VEGF synthesis while tin protoporphyrin (SnPPIX), a potent HO-1 blocker, inhibited H2O2- and hemin-induced VEGF generation. VEGF production was also augmented by platelet-derived growth factor BB (PDGF-BB), which is known to induce HO-1 expression and utilizes H2O2 for its activity. Again, PDGF-BB-induced VEGF synthesis was blocked by SnPPIX. Surprisingly, H2O2 elicited VEGF synthesis in aortic endothelial cells of both wild type and HO-1 knockout mice. In both cell types SnPPIX abolished VEGF production to the same extent. Conclusions: H2O2 is a potent inducer of VEGF synthesis, which occurs concomitantly with enhancement of HO-1 expression. However, the inhibitory effect of SnPPIX, an HO-1 blocker, on VEGF synthesis appears to be unrelated to HO-1 activity. Supported by grants 3 PO4A 049 22 and 6 PO4B 013 from the Polish State Committee for Scientific Research (KBN).

Oral Presentation

39 Are urinary DNA lesions markers of repair? Marcus Cooke, Rosamund Dove, Joseph Lunec, Mark Evans

Oxidative Stress Group, Dept. Clinical Biochemistry, University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom

Oxidative modification of DNA appears to be an inevitable consequence of cellular metabolism, in addition to resulting from exposure to numerous toxic insults. Perhaps as a result, elevated levels of oxidative DNA lesions, such as 8-hydroxy-2’-deoxyguanosine (8-OH-dG), have been measured in numerous pathological conditions [1]. DNA repair is vital in prevent-

ing the persistence of damage, and is therefore apportioned a crucial role in the prevention of disease. Numerous techniques have been applied to the measurement of oxidative DNA damage products in urine, although possible contribution to these levels from the diet and cell turnover has prevented such measurements being wholly attributed to DNA repair. There