Oxidative Stress and Lipid Peroxidation: Physiology and ...Mrs. Vjera Juric and collaborators from the Citizens' Society of Medjimurje Ms. Anja Kresic, Ms. Jelena Zenko. Ms. Silva

Progress Report of the 1. Regional Meeting of the HNE-Club, 7th-8th June 2001, Zagreb, Croatia

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Progress Report of the 1. Regional Meeting of the HNE-Club

Thursday 7th / Friday 8th June 2001 Rudjer Boskovic Institute

Bijenicka 54, HR 10 000 Zagreb, Croatia

Oxidative Stress and Lipid Peroxidation: Physiology and Pathology of 4-Hydroxynonenal

Editors: Neven Zarkovic

Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb

R. Jörg Schaur Institute of Molecular Biology, Biochemistry and Microbiology, Karl Franzen's University, Graz,

Austria

Giuseppe Poli Department of Clinical and Biological Sciences, University of Torino, Torino, Italy

A Network for the Promotion of Research on the Lipid-Derived Aldehyde 4-Hydroxynonenal (HNE)

A Group of Interest within the International Society for Free Radical Research

www.kfunigraz.ac.at/hne-club

Zagreb/Graz, June 2001


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Dedicated to the memories of Hermann and Erwin

Supported by: Croatian Ministry of Science and Technology, Zagreb, Croatia

Rudjer Boskovic Institute, Zagreb, Croatia

Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia

Hrvatsko-austrijsko drustvo za njegovanje kulturnih i gospodarskih odnosa Zagreb, Croatia

Kroatisch-österreichische Gesellschaft zur Förderung kultureller und wirtschaftlicher Bezeihungen, Zagreb, Croatia

Anonymous Sponsor fromGraz, Austria

Acknowledgments: The organizers of the meeting and the editors of the progress report cordially thank for valuable

technical assistance:

Mrs. Vjera Juric and collaborators from the Citizens' Society of Medjimurje

Ms. Anja Kresic, Ms. Jelena Zenko. Ms. Silva Rukavina and

Mr. Tomislav Zarkovic from V. Gimnazija

Mrs. Tea Vukovic and Mr. Goran Goles from Rudjer Boskovic Institute

Edited for the internet by Andreas Jerlich, IMBM, University of Graz


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Some Biomedical Aspects Of Oxidative Stress And 4-Hydroxynonenal

Neven Zarkovic ([email protected])

Rudjer Boskovic Institute, Division of Molecular Medicine, Bijenicka 54, HR-1000 Zagreb, Croatia

Free radicals are atoms or molecules that have unpaired electrons and therefore tend to attack and trap required electron from other, “neighboring” molecules. Consequently, electron “donor” usually becomes unstable and allows the spread of chain reaction of electron removal that will finally end in destruction of bioactive macromolecules (lipids, proteins, and nucleic acids).

Among the aggressive oxygen free radicals, the most reactive and harmful seems to be hydroxyl radical (OH°), which is so reactive that its half-life time is less than a microsecond and his range of activity is within a nm. Such dynamics of interactions between oxygen free radicals and other molecules makes us almost unable to detect their production and interaction with macromolecules in a living system.

However, using sophisticated biophysical methods (such as IR spectroscopy and ESR), biochemical methods of indirect detection of ROS (applying HPLC analysis or chemiluminescence) and finally biological analysis of consequences of ROS interactions with macromolecules (immunodetection of various protein adducts) can give accurate insight into crucial principles by which homeostasis or misbalance of ROS metabolism occur and affect living systems (macromolecules, cells and entire organism).

There are also “defense” molecules, antioxidants, which are able to react with oxygen free radicals and detoxify them at least by turning them into less dangerous molecules, such as hydrogen peroxide, that can again be used to generate new free radicals. Hence, not only oxygen radicals, but also other molecules that do not have unpaired electrons but could easily be changed into free radicals (like in case of Fenton reaction which, in the presence of free iron, leads to production of OH° from hydrogen peroxide) could be dangerous for macromolecules.

Therefore, such substances are denoted as reactive oxygen species (ROS) and are integral part of oxidative stress, defined as a condition of excess in production of oxygen free radicals that exceeds ability of detoxifying mechanisms to prevent damage of the macromolecules caused by harmful reactions of oxygen free radicals.

Oxidative stress is considered as basic pathophysiological mechanism in several chronic and acute disorders, such as atherosclerosis, acute myocardial infarction, brain insult, neurodegenerative disorders, autoimmune diseases, cancer, septic shock, multiple organ failure, etc.

On the other hand, oxidative stress can also be considered as physiological event in case inflammation, immune response to microorganisms, haematoma resorption, traumatic stress and wound healing, etc.

Far less is known about possible beneficial effects of oxidative stress, than about harmful pathologic consequences of excessfull generation of oxygen free radicals. Due to that it is often hard to define when does oxidative stress represent potentially harmful, pathological condition and when it is an integral part of dynamic homeostasis that maintains integrity of the living system.

Lipid peroxidation, which is often developing during oxidative stress, either on the level of cell membranes (not only outer cell membrane, but also within mitochondria and endoplasmic reticulum) or on the level of macromolecules (lipoproteins, in particular low density lipoproteins, LDL) is considered as harmful event that might hardly be reverted and will lead, as a chain reaction, to the spread of oxidative stress.

Producing various ROS in lipid peroxidation (either of saturated or of polyunsaturated fatty acids) will lead to production of the “end products” of lipid peroxidation reactive aldehydes. The most relevant in current biomedicine are malondialdehyde (MDA) and 4-hydroxynonenal (HNE). These aldehydes are able to “simulate” complex oxidative stress and its harmful consequences, hence it is commonly accepted that these molecules are considered as “second toxic messengers” of oxygen free radicals (although they are not ROS).

HNE is especially interesting molecule because it is not produced in detectable amounts not only under oxidative stress, but also in various tissues under physiological conditions, but its physiological role is still not understood. Moreover, HNE has high affinity to bind to proteins making stable protein (peptide) adducts and it seems to have unique feature of retaining its biological activity even if bound to macromolecules and also achieving additional biological activities modifying activities of the macromolecules to which it is conjugated. Because of that it seems likely that HNE might be pluripotent, not only toxic mediator of oxidative stress but might be considered indeed as a “second messenger of oxygen free radicals”. In favor of this are findings of HNE as a growth regulator for normal and malignant cells, proapototic as well as pronecrotic factor that can regulate both cell proliferation as well as differentiation.

Thus, studying physiological and pathological roles of ROS, HNE and related molecules in lipid peroxidation and oxidative stress in general could lead to better understanding of the essential principles of maintaining life under


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hypoxia and reoxigenation, aging of macromolecules (such as LDL) and of entire organism, differences between tissue healing and uncontrolled cell growth in cancer, degenerative diseases and inflammatory response to stress, etc.

These are topics of experimental biochemical (biophysical), biological and medical studies that will be presented in this book, dedicated to great pioneers in this research field, to our dear teachers Prof. Hermann Esterbauer and Prof. Erwin Schauenstein who spent their lives too fast to lead us further in search of life aimed for better understanding of magnificent HNE and its role in homeostasis.

Literature Esterbauer H and Weger W: Ueber die Wirkung von Aldehyden auf gesunde und maligne Zellen, 3. Mitt.: Synthese von homologen 4-Hydroxy-2-alkenalen, II. Monatsh Chem 98: 1994-2000, 1967

Esterbauer H, Zollner H, and Schaur RJ: Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 11: 81-128, 1991.

Zarkovic K, Zarkovic N, Schlag G, Redl H and Waeg G: Histological aspects of sepsis-induced brain changes in a baboon model. In: Shock, Sepsis and Organ Failure, (Schlag G, Redl H and Traber DL, eds), Heidelberg, Springer-Verlag, pp 146-160, 1997.

Kruman T, Bruce-Keller AJ, Bredesen D, Waeg G and Mattson MP: Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis. J Neurosci 17: 5089-5100, 1997.

Dianzani MU: 4-Hydroxynonenal and cell signalling. Free Radic Res 28: 553-560, 1998

Kreuzer T, Grube R, Zarkovic N, Schaur RJ: 4-Hydroxynonenal modifies the effects of serum growth factors on the expression of the c-fos proto-oncogene and the proliferation of HeLa carcinoma cells. Free Radic Biol Med 25: 42-49 1998

Zarkovic N, Zarkovic K, Schaur RJ, Stolc S, Schlag G, Redl H, Waeg G, Loncaric I, Borovic S. Juric G and Hlavka V: 4-Hydroxynonenal as a second messenger of free radicals and growth modifying factor. Life Sci 65: 1901-1904, 1999

Neven Zarkovic: Mechanismus der Tumorentstehung. Pharmazeutishe Zeitung, 145: 239-245, 2000

Borovic S, Meinitzer A, Loncaric I, Sabolovic S, Wildburger R, Tillian M, Martinac P, Stipancic I and Zarkovic N: Monitoring influence of surgical stress on formation of hydroxyl radicals in tumor bearing rats by measuring salicylic acid metabolites. E J Int Fed Clin Chem, 12: 1-4, 2000 http://www.ifcc.org/ejifcc/vol12no2/borovic.htm

Poli G and Schaur RJ: 4-Hyroxynonenal in the pathomechanisms of oxidative stress. IUBMB Life, in press.


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Structural Changes in the Early Oxidation State of LDL Monitored by FT-IR

Greta Pifat-Mrzljak#, Rosana Chehin*, David Rengel*, José Carlos G. Milicua* Félix M. Goñi* and José Luis R. Arrondo+

#Rudjer Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia *Unidad de Biofisica (Centro Mixto CSIC-UPV/EHU),

+Departamento de Bioquímica, Universidad del País Vasco, Apdo. 644, E-48080 Bilbao, Spain

Oxidatively modified low-density lipoprotein (LDL) seems to play a significant role in the initiation and progression of atherosclerosis (Ross, 1993). Metal ions like Cu+2 or Fe+2 are frequently used to initiate LDL oxidation in vitro, since the resulting oxy LDL has similar biological activation to that oxidized in vivo (Esterbauer et al., 1992). The kinetics of Cu+2 induced oxidation is well characterized and different approaches to study oxidation have been used (Meyer et al., 1996, Prassl et al., 1998, etc.).

The question is weather at the beginning of lipid oxidation initial changes in the core lipid can modify apo B conformation before disruption of the lipoprotein particle upon oxidation. These changes at the early stages of copper-mediated oxidation have been monitored by infrared spectroscopy. Previous studies of apo B oxidation by IR (Herzyk et al., 1987) were performed on particles in the advanced stages of oxidation. Variation in native apo B structure has been probed previously (Bañuelos et al., 1995) by the Amide I band. Whatsoever it has been proved that apo B conformation is affected by lipid transitions occurring in the particle lipid core.

In the LDL oxidation process during the lag phase no variation in the structure is observed, indicating that copper binding to the protein does not significantly affect its structure. In the propagation phase, while hydroperoxides are formed but the protein is not derivatized, no changes in secondary structure are produced but the thermal profile of the band corresponding to α-helix is displaced in frequency, indicating changes in tertiary structure associated with this conformation, but not with β-sheet. When aldehyde formation starts a decrease of ca. 3% in the area of bands corresponding to α-helix and β-sheet is produced concomitantly with an increase in β-turns and unordered structure. The two bands corresponding to β-turns vary as well indicating changes in these structures. At this stage the thermal profile also shows variations in frequency for the bands corresponding to α-helix and β-sheet. The results are consistent with the hypothesis that when the polyunsaturated fatty acids from the particle core are modified, this modification is reflected on the surface, in the α-helical components containing the monolayer.

References: Bañuelos, S., Arrondo, J.L.R., Goñi, F.M., Pifat, G. (1995), J. Biol. Chem. 270, 9192-9196.

Esterbauer, H., Gebicki, J., Puhl, H., Jürgens, G. (1992), Free Rad. Biol. Med. 13, 341-390.

Herzyk, E., Lee, D.C., Dunn, R.C., Bruckdorfer, K.R., Chapman, D. (1987), BBA 922, 145-154.

Ross, R. (1993), Nature 362, 801-809.

Meyer, D.F., Nealis, A.S., MacPhee, C.H., Groot, P.H.E., Suckling, K.E., Bruckdorfer, K.R., Perkins, S.J. (1996), Biochem. J. 319, 217-227.

Prassl, R., Schuster, B., Laggner, P., Flamant, C., Nigon, F., Chapman, M.J. (1998), Biochemistry 37, 938-944.


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Inhibitory Effect of Gangliosides on Copper-induced Oxidation of Human LDL and VLDL

V. Lipovac and M. Gavella

University Clinic Vuk Vrhovac, Zagreb, Croatia

Majority of serum gangliosides is from the liver origin and associated with lipoproteins1. Their role in atherogenesis has been proposed, but the mechanism is still not well elucidated2. We have demonstrated that exogenous gangliosides are capable to protect erythrocyte filterability impairment induced by calcium loading3. The aim of this study was to establish the possible influence of exogenous gangliosides on the Cu2+-induced oxidation of LDL and VLDL lipoproteins in vitro. LDL and VLDL were isolated by ultracentrifugation from blood of the healthy donors. Oxidation was performed using 100 µg LDL protein/ml and 5 µM CuSO4, or 25 µg VLDL protein/ml and 17 µM CuSO4 in 0.01 M PBS, pH 7.4, at 30 0C during 300 minutes4,5. Lipoprotein samples were incubated without and with the presence of gangliosides ((Type III or GD1a,; Sigma), at the concentration of 10 and 100 µM. Formation of conjugated dienes were measured at 234 nm. By the analysis of the diene formation kinetics, it was demonstrated that gangliosides protect LDL and VLDL against peroxidation. Lag time duration was prolonged, whereas rate of diene formation was decreased by the presence of gangliosides both in LDL and VLDL oxidation. Present data demonstrate the protective effect of gangliosides against Cu2+-induced peroxidative modification of LDL and VLDL in vitro.

References: Millar JS. The sialylation of plasma lipoproteins. Atherosclerosis 2001; 154: 1-13.

Prokazova NV and Bergelson LD. Gangliosides and atherosclerosis. Lipids 1994; 29: 1-5.

Lipovac V, Gavella M. Rheological action of gangliosides on human erythrocytes Clin Hemorheol. 1994; 14: 347-353.

Esterbauer H, Striegel G, Puhl H, Rothender M. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun. 1989; 6: 67-75.

McEneny J, O, Kane MJ, Moles KW, McMaster C, McMaster D, Mercer C, Trimble ER,Young IS. Very low density lipoprotein subfractions in type II diabetes mellitus: alterations in composition and susceptibility to oxidation. Diabetologia 2000;43:485-493.


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The Fluorescence Spectroscopy of Human Plasma LDL Interaction with Gangliosides

Marina Kveder and Greta Pifat

Rudjer Boskovic Institute, Zagreb, Croatia

ApoB, a constituent of LDL particle, is one of largest monomeric proteins known (4536 amino acid residue). Its interaction with glycolipids is important for LDL interactions in vivo.

Since gangliosides are part of human plasma but they are also associated with LDL, the aim of this study was to investigate their involvement in the oxidative modification of LDL. Using native fluorescence spectroscopy the structural changes of apoB-100 undergoing copper-induced oxidation were followed in the presence and absence of GD1a and gangliosides Type III.

The experimental results indicate a protective role of gangliosides during both the lag phase and propagation phase of the oxidation reaction.


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Pathophysiological Role of Nitric Oxide

Ivan Bilic ([email protected]), Zdenko Kovac

Medical Faculty University of Zagreb Department of Pathophysiology

Nitric oxide (NO) is a free radical molecule enzymatically produced by a number of human cells. It diffuses easily through the tissue, with functional radius of few layers of adjacent cells. Table 1 summarizes relevant biochemical and biophysical NO data. Cell enzymes producing NO are nitric oxide synthases (NOS). NOS use arginine as a substrate to produce free NO and citruline. There are three NOS isoenzymes, two constitutive - eNOS (endothelial), and nNOS (neural) and one inducible form - iNOS. Constitutive NOS forms act in Ca++-dependent manner, whereas iNOS is present and active only after cell stimulation by activators like interleukin 1 (IL-1), tumor necrosis factor α (TNF-α) and lipopolysaccharide (LPS). NO production by cNOS is quantitatively less efficient than that of iNOS. NO is an important biological mediator interacting with wide array of molecular targets (some of them are listed in table 2). NO is involved in different types of biological processes, like cell signalling, cytotoxicity and blood flow regulation.

NO was investigated as the EDRF (endothelium-derived relaxing factor) - substance which mediates endothelial control of vascular tone. However, EDRF was later proven to be identical to NO. Relaxation of smooth muscle cells in blood vessel wall is achieved through NO-mediated stimulation of soluble guanylate cyclase and activation of Ca++-dependent K+-channels. NO synthesis and diffusion from endothelium is stimulated by histamine, bradykinine, acetylcholine, and other factors that activate eNOS by raising intracellular Ca++.

In nervous system NO functions as a neuromodulator. It is produced by post-synaptic neurons excited by glutamate. Back-diffusion to pre-synaptic neurons creates positive feedback loop, which facilitates long-term potentiation of glutamate secretion. It favors neurotransmission through that particular neuronic pathway. This process might be the basis of induction of brain plasticity during learning process. NO also plays the role in neurotransmission within peripheral nerve system in the so-called NANC-nerves (non adrenergic, non cholinergic). It influences some autonomic functions like gastrointestinal motility, penile erection, and urinary bladder relaxation.

Several transcription factors (like NFκB, AP-1) are influenced by NO. Expression of different gene products (including iNOS itself) can thus be driven by NO.

Reaction of NO with superoxide radical produces peroxynitrite (ONOO-), a very reactive compound which can effectively destroy microbial agents. On the other hand, there are several anti-inflammatory NO effects, like free radical scavenging and inhibition of adhesion molecule expression. Those activities contribute to phlogostatic mechanisms responsible for limiting the potentially detrimental effects of inflammation.

NO plays a critical role in the pathogenesis of sepsis. During sepsis, bacterial wall components (LPS, proteoglycan, and lipoteichoic acid) are released into the plasma where they bind to CD14 receptor molecule. Binding of LPS to CD14 is facilitated by LPS-binding protein (LBP, which is one of the acute phase plasma proteins). These interactions activate monocytes, macrophages, and endothelium. Several autostimulatory feedback cytokine loops (IL-1, TNF-α) can enhance activation, leading to excessive stimulation of iNOS expression. As a consequence, massive production of NO takes place, inducing ubiquitous vasodilatation and hypotension. Hence, vasohypotonic circulatory shock occurs, leading to multi-organ dysfunction syndrome (MODS) and multi-organ failure syndrome (MOFS). Further progression very often leads to death. At the cellular level, oxygen utilization and energy metabolism appear to be severely compromised, but cytosolic ATP concentration is maintained for a long period. However, cells appear to be unable to use energy and develop dysfunction and failure. All these processes can cause hypofunction or afunction of different organ systems (most frequently kidney, lung and liver). Many macromolecular nitrosylations due to NO overproduction have been well documented in sepsis. However, their real impact on pathophysiology of the syndrome remains uncertain.

NO appears to have cytotoxic effector function. It is achieved through multiple molecular mechanisms. Due to its interaction with enzymes which are responsible for cell energy production (mitochondrial complex I and II, aconitase), NO can deplete cell energy stores. This can be achieved also indirectly, through activation of ADP-ribosylation, which tends to exhaust cellular NADPH. Peroxynitrite acts as non-specific agent of cellular damage. It can irreversibly alter lipids, proteins and DNA. Consequent lipid peroxidation produces secondary mediators of cytotoxicity (HNE, MDA etc.). The ultimate consequence of these NO effects may be cell death. Role of NO as cell death mediator is well documented during the inflammation and glutamate excitotoxicity in CNS (caused by stroke, trauma, neurodegenerative disorders, etc.).

The pathogenesis of atherosclerosis involves NO as the contributory mechanism. Oxidatively modified LDL, which is a potent atherogenic substance, can be generated by peroxynitrite. Peroxynitrite is produced by inflammatory cells, which


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infiltrate the blood vessel wall. Moreover, atheroslerosis-affected blood vessels seem to be deprived of normal protective NO effects. They tend to narrow due to impaired vasodilatation and increased platelet aggregation.

Present understanding of physiological and pathophysiological roles of NO points out that its systemic biological actions are the result of many autacoid as well as toxic processes. Those activities are driven by tissue NO concentration along with the multiple microenvironmental factors like oxidative stress, cell type, and availability of protein targets or regulators.

Table 1. Biochemical and biophysical characteristics of nitric oxide Molecular mass 30,006 N -- O bond distance 1,1508 angstrom Saturated solution of NO ≈ 3 mM Main products of redox reactions* Nitrosonium ion, nitroxyl anion, nitrates, nitrites Steady-state tissue concentration 10 nM - 1 µM Diffusion constant in aqueous solution at 37°C # 3300 µm2s-1

*Depending on the redox microenvironment, NO can exert oxidative or reductive effects. #About 1,5 times higher diffusibility compared to molecular oxygen.

Table 2. Mechanisms of NO-mediated pathophysiological processes Protein targets of nitric oxide Immediate consequence Effects at the cellular level Guanylate cyclase ACT cGMP rise

Smooth muscle relaxation K+

Ca channel ACT Hyperpolarisation Mitochondrial oxidoreductases - complex I and II INH Inhibition of mytochondrial

Energy depletion Aconitase INH respiration AP-1 ACT/INH*

Transcription modification

Triggering of the cell signaling cascade

NFκB ACT/INH*

Abbreviations: ACT - NO activates function of a named molecule, INH - NO inhibits molecule.

AP-1 - activator protein 1 - transcription factor composed of c-jun i c-fos sub-units. It is involved in control of both basal and inducible genes; NFκB - nuclear factor κ B - transcription factor responsible for inducing many different genes during inflammation.

*NO mediated effects on transcription vary according to cell type and microenvironmental conditions.

Literature Mori M, Gotoh T: Regulation of nitric oxide production by arginine metabolic enzymes. Biochem Biophys Res Comm 2000; 275: 715-9

Kelm M: Nitric oxide metabolism and breakdown. Biochim Biophys Acta 1999; 1411: 273- 89

Nitric oxide, chapter in Vincent JL ed. Yearbook of Critical Care and Emergency Medicine. Berlin - Heidelberg: Springer Verlag;1997, pg. 197-239

Koprowski H, Maeda H, ed. The role of nitric oxide in physiology and pathophysiology. Curr Top Micro Immunol 1995; 196:

Moncada S et al.: The L-arginine - nitric oxide pathway. New Engl J Med 1993; 329: 2002- 2012


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Effects of 4-Hydroxynonenal and Splenic Cells on Rat Liver Cells In Vitro

Borovic S.1, Cipak A.1, Zarkovic N.1, Scukanec M.2

1 Rudjer Boskovic Institute, Division of Molecular Medicine, Zagreb, Croatia 2 Division of Pathology, Faculty of Medicine, Zagreb, Croatia

Introduction

Oxidative stress causes lipid peroxidation generating a great diversity of aldehydes called “second toxic messengers of reactive oxygen species”, due to their high reactivity with macromolecules and toxicity for living cells. One of the most intensively studied aldehyde is 4-hydroxynonenal (HNE). Previous studies identified HNE as highly cytotoxic aldehyde, although it can be found in physiological conditions in cells and tissues of human and animal origin (Esterbauer et al., 1991, Schaur and Poli, 2000). Oxidative stress was shown to play a regulatory role in liver regeneration (Okiawa and Novikoff, 1995). During liver regeneration migration of lymphocytes from spleen to liver also seems to play a certain regulatory role. Namely, splenic cells excrete cytokines that additionally activate cytotoxic cells, which kill altered or damaged liver cells during regeneration (Suzuki et al., 1996). The aim of this study was to investigate if HNE-mediated oxidative stress can influence interactions between liver cells and splenic cells.

Materials and methods

Primary liver cell culture was established after isolating liver from adult male Wistar rats. Interactions between spleen and liver cells in the condition of HNE-mediated oxidative stress were measured by the 3H-thymidine assay at 4th day. Liver cells were precultured for 30 minutes in medium with 5% fetal calf serum (FCS) and with 100 µM HNE (controls did not have HNE in medium) before cultivation with spleen cells. The ability of spleen cells to proliferate was measured in the presence of liver cells (non-treated and HNE-pretreated) destroyed with ultrasound, bovine serum albumin (BSA; alone or preincubated with HNE) and plant mitogens (phytohemagglutinin, PHA and concanavalin A, ConA). The Trypan blue exclusion assay was used to monitor viability and growth of liver cells, normal or damaged by cytotoxic concentration (100 µM) of HNE; and splenic cells for 4 days.

All experiments were preformed in triplicates. Significance was calculated according to the Student’s t-test, value of p<0,05 was considered as significant.

Results

Mixed cultures of liver and splenic cells showed decreased 3H-thymidine incorporation in comparison to the liver cells cultured without splenic cells (p<0,05). Opposite to that, mixed cultures of splenic cells and liver cells pretreated with cytotoxic concentration of HNE showed increased incorporation of 3H-thymidine (p<0,05) (Figure 1). Liver cells destroyed with ultrasound (non-treated and HNE pretreated) and BSA (alone and HNE-preincubated) did not stimulate proliferation of splenic cell (Figure 2).

Figure 1. Mixed culture of spleen and liver cells – 3H-thymidine assay Mixed cultures were cultivated in medium with 5% of FCS. Liver cells were pretreated with cytotoxic (100µM) concentration of HNE for 30 minutes. Expected value was calculated as sum of 3H-incorporation in individual cultures of liver and spleen cells. Obtained value was divided by expected value and expressed as percentage. FCS = fetal calf serum; HNE = 4-hydroxynonenal


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Figure 2. Influence of lyzed liver cells (non-treated and HNE pretreated) and BSA (alone and HNE-preincubated) on spleen cells (controls were spleen cells cultivated alone) – 3H-thymidine assay Liver cells were incubated in medium with 5% of FCS without or with addition of cytotoxic concentration of HNE for 30 minutes. Afterwards, liver cells were lysed with ultrasound. Concentration of BSA was adjusted to be the same as concentration of proteins in lysed liver cells. BSA = bovine serum albumin; FCS = fetal calf serum; HNE = 4-hydroxynonenal

Plant mitogens ConA and PHA caused spleen cell proliferation (p<0,05) (Figure 3). Trypan blue exclusion assay showed that the number of viable spleen cells was decreasing in culture during 4 days (1st to 4th day, p<0,05). The assay also showed that the number of viable liver cells was increasing (1st to 4th day, p<0,05). Thus, liver cells damaged by HNE retained their viability if cultured with splenic cells; otherwise they died (Figure 4).

Figure 3. Reactivity of spleen cells on plant mitogens, phytohemagglutinin (PHA) and concanavalin A (ConA)– 3H-thymidine assay

Figure 4. Influence of spleen cells on liver cells – Trypan blue exclusion assay Liver cells (non-treated and HNE-pretreated) were cultured either alone or with spleen cells in ratio 1:40. HNE = 4-hydroxynonenal.


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Discussion

We expected a decrease in 3H-thymidine incorporation in mixed cultures of HNE-damaged liver cells and spleen cells that would reflect a cytotoxic effect of HNE combined with a cytotoxicity of splenic cells. Hence, to investigate if the unexpected increase of 3H-thymidine incorporation was due to the proliferating spleen cells, these cells were additionally cultured with liver cells destroyed with ultrasound. However, lysates prepared from non-treated liver cells or those pretreated with HNE did not stimulate proliferation of splenic cells although ConA and PHA induced a mitogenic response of the splenic lymphocytes.

The Trypan blue exclusion assay showed that liver cells damaged by HNE recovered on the 4th day of the experiment (the day when the 3H-thymidine assay was performed) but only if they were cultivated together with splenic cells. Thus, we can conclude that splenic cells secrete some factors or on some other way support recovery of HNE-damaged liver cells.

References 1. Esterbauer H, Zollner H, Schaur RJ: Chemistry and biochemistry of 4-hydroxynonenal, malonaldahyde and related aldehydes. Free Radic Biol Med 11: 81-128, 1991.

2. Poli G, Schaur RJ: 4-Hydroxynonenal in the pathomechanisms of oxidative stress. IUBMB Life, 50, 1-7, 2000.

3. Okiawa I, Novikoff PM: Catalase negative peroxisomes: transient appearance in rat hepatocytes during liver regeneration after partial hepatectomy. Am J Pathol 146: 673-686, 1995.

4. Suzuki S, Nakamura S, Serizawa A, Sakaguchi T, Konno H, Muro H, Kosugi I, Baba S: Role of Kupffer cells and the spleen in modulation of endotoxin-induced liver injury after partial hepatectomy. Hepatol 24: 219-325, 1996.


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Oxysterols of Biological Relevance and Their Involvement in the Progression of the Fibrotic Plaque

Giuseppe Poli and Gabriella Leonarduzzi

Dept. of Clinical and Biological Sciences, University of Torino at the S. Luigi Battista Hospital, Torino, Italy

Deposition of blood cholesterol in the subendothelial space of major arteries is the recognized main feature of the fibrotic plaque as well as a key event in the progression of the atherosclerotic disease process (1,2). However, the mechanisms by which cholesterol triggers and sustains the fibrotic degeneration of blood arteries remains undefined.

For a proper insight into this problem, useful appears to consider cell interactions involved in the formation of the fibrotic plaque as well as the relative molecular mediators. It is now generally accepted that, in the different organs undergoing fibrosclerotic transformation two types of cells act as key-players, namely activated macrophages and fibroblasts or fibroblast-like cells (3,4). The latter definition is certainly appropriate in the genesis of the atheromasic lesion of medium and high size arteries, where mainly smooth muscle cells are “induced” to acquire the phenotype and function of fibroblasts.

The cross-talk between macrophages and fibroblasts or smooth muscle cells is achieved through the expression of a number of inflammatory cytokines, like platelet-derived growth factor (PDGF), tumor necrosis factor α (TNFα), interleukin 1 (IL-1), but in particular, by transforming growth factor β1 (TGFβ1), by far the strongest profibrogenic cytokine (5,6). In relation to this point, membrane lipid peroxidation appears one likely candidate for effective modulation of TGFβ1 levels in the arterial wall. We previously demonstrated that 4-hydroxynonenal (HNE), a major aldehydic end-product of peroxidation of ω-6-fatty acids (7), is able to markedly up-regulate both expression and synthesis of TGFβ1 in macrophages (8). As for the phagocytes found in the arterial wall, an “activating” stimulus may be provided by oxidized low density lipoproteins (oxLDLs). In addition to the well documented formation of HNE in LDL lipids (7), oxLDLs contain variable amount of oxysterols, i.e. 27-carbon products of cholesterol oxidation, which become quantitatively important in hypercholesterolemic subjects (9-12). Oxysterols have been shown to exert a great number of biochemical effects (13,14), but their potential action as lipid oxidation products on the expression of fibrogenic cytokines by macrophages was not investigated until recently, when we demonstrated a strong profibrogenic stimulus exerted by oxysterols in amounts comparable to that found in hypercholesterolemic blood (15). Uptake of these oxysterols by macrophages up-regulated expression and synthesis of TGFβ1 in these cells. Such experimental evidence results indicate a possible mechanistic link between increased cholesterol in plasma and arteries and stimulated fibrogenesis. Uptake of cholesterol by macrophages gives rise to foam cells as characteristic components of an atheroma, but cholesterol alone does not stimulate fibrogenic signaling from these phagocytes, whereas its various oxidation products are highly effective. Since deposition of oxidized LDL into the arterial wall and its uptake by vascular cells is thought to be an early atherogenic event, the delivery of oxysterols via oxidized LDL may contribute to specific steps in the elaboration of atherosclerosis lesions. The marked accumulation and enrichment of oxysterols in the subendothelial space not only reflects an oxidative burden but may stimulate key events in the progression to a fibrotic lesion.

References Ross, R. (1993) The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362, 801-809

Napoli, C., D’Armiento F.P., Mancini, F.P., Postiglione, A., Witztum, J.L., Palumbo, G., and Palinski, W. (1997) Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia. Intimal accumulation of low density lipoprotein, its oxidation precedes monocyte recruitment intoearly atherosclerotic lesions. J.Clin.Invest. 100, 2680-2690

Poli, G. (2000) Pathogenesis of liver fibrosis: role of oxidative stress. Molec. Aspects Med. 21,49-98

Poli, G., and Parola, M. (1997) Oxidative damage and fibrogenesis. Free Radical Biol. Med.22, 287-305

Kovacs, E. J. (1991) Fibrogenic cytokines: The role of immune mediators in the development of scar tissue. Immunol. Today 12, 17-23

Grande, J.P. (1997) Role of transforming growth factor β in tissue injury and repair. Proc. Soc. Exp. Biol. Med. 214, 27-40

Esterbauer, H., Schaur, R.J., and Zollner, H. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biol. Med. 11, 81-128

Leonarduzzi, G., Scavazza, A., Biasi, F., Chiarpotto, E., Camandola, S., Vogl, S., Dargel, R., and Poli, G. (1997) The lipid peroxidation end-product 4-hydroxy-2,3-nonenal up-regulates transforming growth factor β1 expression in the macrophage lineage: a link between oxidative injury and fibrosclerosis. FASEB J. 11, 851-857

Hodis, H. N., Crawford, D. W., and Sevanian, A. (1991) Cholesterol feeding increases plasma and aortic tissue cholesterol oxide levels in parallel: further evidence for the role of cholesterol oxidation in atherosclerosis. Atherosclerosis 89, 117-12

Hodis, H. N., Kramsch, D.M., Avogaro, P., Bittolo-Bon, G., Cazzolato, G., Hwang, J., Peterson, H., and Sevanian A. (1994) Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL-). J. Lipid. Res. 35, 669-677


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Dzeletovic, S., Breuer, O., Lund, E., and Diczfalusy, U. (1995) Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry. Anal. Biochem.225, 73-80

Chang, Y. H., Abdalla, D. S., and Sevanian, A. (1997) Characterization of cholesterol oxidation products formed by oxidative modification of low density lipoprotein. Free Radical Biol. Med. 23, 202-214

Brown, A. J., and Jessup, W. (1999) Oxysterols and atherosclerosis. Atherosclerosis 142, 1-28

Schroepfer, G.J. (2000) Oxysterols: modulator of cholesterol metabolism and other processes. Physiol. Rev. 80, 361-554

Leonarduzzi, G., Sevanian, A., Sottero, B., Arkan, M. C., Biasi, F., Chiarpotto, E., Baþaða, H. and Poli, G. (2001) Up-regulation of the fibrogenic cytokine TGFβ1 by oxysterols: a mechanistic link between cholesterol and atherosclerosis. FASEB. J., in press (July issue).


15

Proliferation and Apoptosis of Vascular Smooth Muscle Cells – Role of Oxidized Phospholipids and HNE

A. Moumtzi and A. Hermetter

Department of Biochemistry, Technical University of Graz,

Petersgasse 12/II, A-8010 Graz, Austria

Migration, proliferation and death of smooth muscle cells (SMC) in the intima of arterial walls are characteristics typical of atherosclerotic lesions. Oxidative modification of low density lipoprotein (LDL) is thought to play a major role in this respect, because it renders LDL atherogenic. The pathophysiological properties of oxidized LDL (oxLDL) may be attributed in part to their content of biologically active (phospho)lipid fragments. For instance, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 4-hydroxy-2-nonenal (HNE) have been identified as components of oxLDL and have been found in atherosclerotic plaques.

We investigated the role of these lipid oxidation products on induction of proliferation and apoptosis in cultured human and rat aortic SMC. Cell proliferation was determined from the incorporation of BrdU into the cellular DNA. Apoptotic cells were detected by YOPRO-staining. The cellular responses towards the oxidized phospholipids depended on dose (0,1-10µM), culture conditions and the cell line used. POVPC and PGPC stimulated proliferation of rat SMC. Under the same conditions apoptotic rates were similar to the controls. The same phospholipids did not affect very much cell growth of human SMC. At doses above 4µM, HNE inhibited growth of both cell lines. In the same concentration range it induced apoptosis of rat SMC.

Very little is known about the site of action of the oxidized phospholipids. Identifying their intracellular destination and distribution would be a decisive step towards a better understanding of activated signal transduction cascades. Fluorescence microscopy experiments with a fluorescent, pyrene-labeled analogue of PGPC showed that this compound was rapidly taken up by rat aortic SMC and became distributed over the whole cell except the nucleus.

These data support the assumption that the respective lipid is not only available in the plasma membrane but also inside the cells within rather short time. Thus, it is likely to interact directly with intracellular components of signal transduction leading to the observed effects on proliferation and apoptosis of SMC.


16

Effects of 4-Hydroxynonenal and Hypoxia on Brain Endothelial Cells

P. M. Eckl*, G. M. Karlhuber* and H.C. Bauer**

*Institute of Genetics and General Biology, University of Salzburg, Hellbrunnerstr. 34, A-5020 Salzburg, Austria

**Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstr. 11, A-5020 Salzburg, Austria

In higher vertebrates, most parts of the central nervous system (CNS) are separated from the bloodstream by the blood-brain barrier (BBB) represented by the cerebral microvascular endothelial cells (cECs) (1). The BBB is responsible for the selective uptake of ions and nutrients guaranteeing the homeostatic environment for the proper functioning of the CNS. Endothelial cells grow in general in epithelial monlayers and are thus exposed to various endogenous and exogenous toxic agents. Reactive oxygen metabolites, such as the hydroxyl radical, the superoxide radical, or hydrogen peroxide (H2O2) are produced in the course of metabolic activities in the brain (2). Under physiological conditions, their action is minimized and compensated by antioxidative mechanisms (3). Disturbances of the balance between oxidative and antioxidative processes appear to have a special significance for cell and tissue injury, which may result in a breakdown of the BBB (3).

Increasing evidence indicates that brain tissue injury after ischaemia is related to the production of oxygen free radicals. The injury of organs and tissues after ischemia causing the interruption of oxygen supply, results very likely from oxygen free radicals which are released during the period of reperfusion. Recent advances in understanding the fundamental mechanisms of post-ischaemic injury have suggested that most tissue injury is associated with admission of oxygen to the tissue at the time of reperfusion (4,5).

Free radicals released during reperfusion are able to initiate lipid peroxidation which generates a large variety of water-soluble carbonyl compounds, such as malondialdehyde, 2-alkenals and 4-hydroxyalkenals (6,7). Due to their high reactivity, 4-hydroxyalkenals appear to be the biologically most significant class of peroxidation products (8).

In order to study both the effect of hypoxia/reoxygenation and that of hydroxyalkenals on cerebral endothelial cells we have performed two sets of experiments. We focused our investigations a) on the use of 4-hydroxynonenal, the major 4-hydroxyalkenal produced in biological membranes, and that has been demonstrated to exert cyto- and genotoxic effects at concentrations between 0.1 and 100 µM (9-11) and b) on exposure of the cells to hypoxic conditions and subsequent reoxygenation.

For these experiments, cloned cECs were used, which reversibly develop into two different phenotypes, depending on the culture conditions. When endothelial cell growth supplement (ECGS) and heparin are added to the culture medium, cECs exhibit an epitheloid appearance (type I cECs), whereas cells grown without these substances appear elongated and spindle-shaped (type II cECs). Both phenotypes differ in their proliferation rate and show a different protein pattern and expression of endothelial cell-specific macromolecules. BBB-associated marker enzyme activities in cultured cECs, like (gamma-Glutamyl-Transpeptidase and Na+ K+-ATPase, are elevated in type I cells, whereas the expression of smooth muscle actin and increased cell migration are typical of type II cells (12,13). Both phenotypes therefore most likely represent the two major functions of EC in the brain: the BBB function and the neovascularization and capillary formation. Thus, these cloned cerebral endothelial cells appear to be a suitable in vitro model to investigate cyto- and genotoxic effects of oxidative stress in the brain.

References: M.W.B. Bradbury, The Concept of a Blood-Brain Barrier, Wiley and Sons, New York, 1979.

T. Matsuyama, Free radical-mediated cerebral damage after hypoxia/ischemia and stroke, in: G. J. Ter Horst, J. Korf (Eds.), Clinical Pharmacology of Cerebral Ischemia, vol. 7, Humana Press, Totowa, 1996, pp. 153-184.

L. Betz, Oxygen free radicals and the brain microvasculature, in: W. M. Pardridge (Ed.), The Blood Brain Barrier, Raven Press, New York, 1993, pp. 303-321.

J.M. McCord, R.S. Roy, The pathophysiology of superoxide: roles in inflammation and ischemia, Can. J. Physiol. Biol. Med. 2 (1982) 325-345

L. Ernster, Biochemistry of reoxygenation injury, Crit. Care Med. 16 (1988) 947-953.

Benedetti, A. F. Casini, M. Ferrali, M. Comporti, Extraction and partial characterization of dialysable products originating from the peroxidation of liver microsomal lipids and inhibiting microsomal glucose-6-phosphatase activity, Biochem. Pharmacol. 28 (1979) 2909-2918.

H. Esterbauer, Aldehydic products of lipid peroxidation, in: D. C. H. McBrien, T. F. Slater (Eds.), Free Radicals, Lipid Peroxidation and Cancer, Academic press, London, 1982, pp. 101-128.

H. Esterbauer, R. J. Schaur, H. Zollner, Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes, Free Radic. Biol. Med. 11 (1991) 81-128.


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P. Eckl, H. Esterbauer, Genotoxic effects of 4-hydroxyalkenals, Adv. Biosci 76 (1989) 141-157.

H. Esterbauer, P. M. Eckl, A. Ortner, Possible mutagens derived from lipids and lipid precursors, Mutation Res. 238 (1990) 223-233.

P. M. Eckl, A. Ortner, H. Esterbauer, Genotoxic properties of 4-hydroxyalkenals and analogous aldehydes, Mutation Res. 290 (1993) 183-192.

H. C. Bauer, U. Tontsch, A. Amberger, H. Bauer, (gamma-Glutamyl-Transpeptidase (gGTP) and Na+K+-ATPase activities in different subpopulations of cloned cerebral endothelial cells: responses to glial stimulation, Biochem. Biophys. Res. Commun. 168 (1990) 358-363.

Amberger, P. F. Lemkin, P. Sonderegger, H. C. Bauer, ECGF and heparin determine differentiation of cloned cerebral endothelial cells in vitro, Mol. Chem. Neuropathol. 20 (1993) 33-43.


18

Mechanisms of Iron-Induced Modifications of Creatine Kinase in Rat Brain In Vitro: Possible Involvement of 4-Hydroxynonenal

Horakova L.*, Ondrejickova O.*, Vajdova M.*, Korytar P.**, Durackova Z.** and Schaur R.J.***

* - Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dubravska 9, 842 16, Bratislava, Slovakia ** - Department of Medical Chemistry and Biochemistry, Medical Faculty, Comenius University, Sasinkova 2, 813 72

Bratislava, Slovakia *** - Institute of Molecular Biology, Biochemistry and Microbiology, Karl-Franzen's University, A 8010 Graz, Austria

Oxidative modification of proteins occurs in cells during oxidative stress in various pathological states as well as aging, and it may include primary and secondary mechanisms. The most prominent mechanisms of primary modification are those involving site–specific metal-ion-catalyzed reactions. His, Arg, Lys, Pro, Met, Cys residues are among the most common sites oxidized by metal catalyzed oxidation systems (Stadtman, 1993).

It is believed that the Fe(II) binds to a metal binding site on the protein and then the protein-Fe(II) complex reacts with H2O2 to generate in situ an activated oxygen species (·OH, ferryl compounds), which further reacts with the side chains of amino acid residues at the metal binding site. So free radical species directly attack amino acids converting them to carbonyl groups (Levine, 1983; Davies et al.,1999).

Secondary modifications occur when proteins are modified by molecules generated by oxidation of other molecules, for example by 4-hydroxynonenal (HNE) produced by end products of lipid peroxidation (Esterbauer et al., 1991). It was demonstrated that histidine, cysteine and lysine residues of proteins are important targets for modification by HNE (Uchida and Stadtman, 1992; Reinheckel et al., 1998; Eaton et al., 1999). HNE has a considerably longer half-life than free radical species and is capable of inhibiting the function of key enzymes (Siems et al., 1996).

We studied oxidative modification of creatine kinase (CK) in rat brain in vitro where oxidative stress was induced by in vitro addition of Fe/ascorbate. CK belongs to the enzymes sensitive to metal catalyzed oxidative modification (Fucci et al., 1983; Levine, 1983; Aksenov et al., 1997). We analyzed the potential involvement of individual reactive oxygen species in oxidative modification of CK and tried to establish whether primary or secondary, or both mechanisms of modification were included in oxidative stress induced in rat brain in vitro.

The site-specific nature of pure CK modified by metal catalyzed oxidation has been established (Fucci, 1983). This type of modification is usually indicated by the observation that the inactivation of enzymes is relatively insensitive to the protection by free radical scavengers (OH radical scavenger), as e.g. mannitol. An involvement of superoxide anion in this modification was excluded as SOD failed to inhibit the metal-catalyzed oxidative modification (Table 1). Neither mannitol nor SOD prevented oxidative modification of CK in rat brain homogenates oxidized by Fe/ascorbate in our experiments.

Involvement of H2O2 in this oxidative reaction was indicated by the finding that the oxidative modification of CK in our experiments was inhibited by catalase, which is in agreement with the results of Fucci et al., (1983) on pure CK. Glutathione strongly prevented a decrease in CK activity in our experiments. That could be due to the rapid consumption of GSH via glutathione peroxidase reaction, though also to the high reactivity of HNE generated from lipids during oxidation with sulfhydryl groups. Glutathione easily reacts with HNE (Esterbauer et al., 1991) and enzymes inactivated by HNE can be reactivated by excess glutathione. Further, GSH is an OH radical scavenger, yet it is known that OH radicals are not involved in metal catalyzed oxidative modification, as confirmed also in our experiments where mannitol, a known OH radical scavenger, did not influence CK activity.

Antioxidant Control

CK(U/mgprot.) Oxidation Antioxidant Loss of CK activity

in percentage* Catalase (49 000 U/ml) 169.9±9.6 84.3±5.5 128.3±23.8 49

SOD (630 U/ml) 174.3±12.0 92.5±2.0 66.2±2.4 132 EDTA (200 µM) 177.8±2.5 92.5±2.0 39.4±1.0 162 Manitol (30 µM) 209.1±1.2 137.7±0.4 139.8±3.5 97

Manitol (100 µM) 209.1±1.1 137.7±0.4 120.6±5.6 124 Glutathione (30 µM) 213.4±2.2 140.5±2.0 181.0±1.7 44.4

Glutathione (100 µM) 213.4±2.2 140.5±2.0 192.9± 5.6 28.1

Table 1. Effects of antioxidants and antioxidative enzymes on oxidatively modified CK in rat brain homogenates. The results are expressed as mean ± SEM. The difference in the activity of CK in the control and oxidized samples was set as 100 percent loss of CK activity in the absence of antioxidant. * -100 % injury reflects values obtained if respective antioxidants were not used (to compare effects of different antioxidants performed in different experiments).


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To establish whether and at which concentrations CK may be oxidatively modified by HNE, pure CK was incubated in the presence of 10 and 64 µmo/l of HNE for 30 min at 37°C. The activity of CK incubated with HNE decreased significantly (Table 2). Simultaneously, the protein carbonyls, determined by electrophoresis and immunoblotting, increased at 10 µmol/l HNE or disappeared, probably due to crosslinking of CK at 64 µmol/l HNE.

Incubation: 30 min Incubation: 120 min CK U/mg prot.

CK 142.6±5.9 159.4±14.2 CK+HNE (10 µM) 108.6±6.2 (76.1%) 38.8±0.3 (21%)

CK 204.8±4.7 220.3±5.0 CK+HNE (64 µM) 43.2±2.9 (24.3 %) 0 (0%)

Table 2. Effect of HNE on CK Results are expressed as mean ± SEM.

The concentration of HNE in rat brain homogenates after oxidative stress (Table 3) was determined by HPLC and was in the range of 10 – 16 nmol/mg prot., corresponding to a concentration of 10-16 µmol/l HNE. This indicates that CK of rat brain homogenates oxidized by Fe/ascorbate may be impaired not only directly by oxygen radicals but also secondarily by HNE.

Time of incubation (min): 0 10 20 30

HNE (nmol/mg prot. corresponding µM) 16.05±0.62 10.03±0.25 10.06±0.98 12.17±0.31

Table 3. HNE concentration in rat brain homogenates in the presence of Fe/ascorbate All values are expressed as mean ± SEM.

References: Stadtman ER. Oxidation of free amino acids and amino residues in proteins by radiolysis and by metal-catalyzed reactions. Annu Rev Biochem 1993; 62: 797.

Levine RL. Oxidative modification of glutamine synthetase. J Biol Chem 1983; 258: 11823.

Davies M.J, Fu S, Wang H, Dean RT. Stable markers of oxidant damage to proteins and their application in the study of human disease. Free Rad Biol Med 1999;27: 1151.

Esterbauer H, Schaur RJ., Zollner H. Chemistry and biochemistry of 4- hydroxynonenal, malondialdehyde and related aldehydes. Free Rad Biol Med 1991; 11: 81.

Uchida K, Stadtman E.R. Modification of histidine residues in proteins by reaction with 4-hydroxynonenal. Proc Natl Acad Sci USA 1992; 89: 4544.

Reinheckel T, Noack H, Lorenz S, Wiswedel I, Augustin W. Comparison of protein oxidation and aldehyde formation during oxidative stress in isolated mitochondria. Free Rad Res 1998. 29, 297-305.

Eaton P, Li JM., David J, Shattock H, Shattock M. Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart. Am J Physiol 1999; 276: H935.

Siems, W.G., Hapner, S. J., Kuijk, F.J.G.M. 1996. 4-Hydroxynonenal inhibits the Na+-K+-ATPase. Free Radic. Biol. Med. 20: 215-223.

Fucci L, Oliver CN, Coon MJ, Stadtman ER. Inactivation of key metabolic enzymes by mixed-function oxidation reactions: Possible implication in protein turnover and aging. Proc Natl Acad Sci 1983; 80:1521.

Aksenov MY, Aksenova MV, Payne RM, Smith CD, Markesbery WR, Carney JM. The expression of creatine kinase isoenzymes in neocortex of patients with neurodegenerative disorders: Alzheimer´s and Parkinson´s disease. Exp Neurol 1997;142: 458.


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Apoptosis in HNE Lesioned Neurons and the Use of HNE as a Marker of Oxidative Stress in Models for Age Related Diseases

Robert Wronski, Nicole Golob, Susanne Kronawetter and Manfred Windisch

JSW Research Rankengasse 28 8020 Graz, Austria, http://www.jswresearch.com

We used primary cultured cortical chicken neurons to investigate the effects of HNE (4-hydroxynonenal) on the occurrence of apoptosis in vitro. Viability, caspase 3 activity and changes in membrane permeability were assessed in the current experiments. Two different viability assays, the MTT and the LDH assay, were applied in parallel to obtain more reliant results. Caspase 3 activity was measured in the cell lysates using the fluorescence labeled substrate Ac-DEVD-AMC.

Apoptosis specific membrane permeability changes occur rather late in the time course of apoptosis. These alterations were observed using the fluorescent DNA intercalating dyes propidium iodide and YOPRO 1.

The MTT as well as the LDH assay lead to corresponding results revealing a clear cytotoxic effect in our model.

Levels of caspase 3 activity were elevated upon the HNE lesion. The experiments with the two DNA staining dyes revealed HNE to increase apoptosis as well as necrosis, whereas the necrosis signal was pronounced more clearly.

Since the membrane alterations are a sign of late apoptosis it appears likely, that HNE induces apoptosis, which turns into secondary necrosis.

This could be due to an HNE related misfunction of essential elements in the apoptotic pathway after HNE mediated induction of apoptosis.

In the near future we intend to determine the extent of oxidative stress in some of our disease related cell culture models. JSW-Research has established a transgenic cell line expressing parkinsonian α-synuclein. Recent findings revealed this protein to be strongly involved in radical induced cellular stress. Consequently a relevant assessment of that form of cell stress in these and other cells appears to be essential. For that reason it is our plan to use HNE adducts as quantifiable markers for oxidative stress.


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Pathomorphological Distribution of HNE-Protein Adducts in Brain

Kamelija Zarkovic1, Gordana Juric1, Iva Loncaric1, Georg Waeg2, Heinz Redl3, Svorad Stolc4 and Neven Zarkovic5

1 - Division of Neuropathology, Medical Faculty, Clinical Hospital Center Zagreb, Zagreb, Croatia 2 - Institute of Molecular Biology, Biochemistry and Microbiology, Graz, Austria

3 - Ludwig Boltzman Institute of Experimental and Clinical Traumatology, Vienna, Austria 4 - Institute of Experimental Pharmacology, Slovak Academy of Science, Bratislava, Slovakia

5 - Rudjer Boskovic Institute, Division of Molecular Medicine, Zagreb, Croatia

Introduction

Aldehyde 4-hydroxynonenal (HNE) is a lipid peroxidation product generated during pathophysiological conditions based on the production of reactive oxygen species (ROS). ROS induce peroxidation of the lipids (cellular membrane lipids i.e. polyunsaturated fatty acids - PUFAs or circulating lipoprotein molecules) generating highly reactive aldehydes, which are considered as ”second toxic messengers” of free radicals (1, 2). Thus produced aldehydes are very toxic and damage tissue. One of the most important products of lipid peroxidation is highly reactive aldehyde 4-hydroxynonenal (HNE) (2, 3). There are data indicating involvement of HNE and related aldehydes in ROS induced damage of various organs, particularly brain, after different pathological events. The Brain is inherently vulnerable to damage mediated by ROS because of strong oxidative metabolism and high intracellular production of superoxide in oxygen mitochondrial electron transport. Brain has very limited ability to conduct anaerobic glycolysis and so it is vulnerable to hypoxia. Production of superoxide by mitochondria increases dramatically in hypoxia. High iron concentration and high lipid to protein ratio in brain tissue in contrast to relatively low levels of antioxidant (glutathion and glutathion related enzyme) increases brain tissue vulnerability to oxidative stress (4). Hence, morphological evaluation of the tissue and cellular distribution of the mediators of oxidative stress that would show probable differences between the different types of cells or parts of organs involved in pathological events were not possible, because radicals are so reactive that live short before interact with another molecule. Specific monoclonal antibodies were developed against HNE-protein (or peptide) conjugate(s) that allow morphological analysis of the tissue distribution of the aldehyde (5). These monoclonal antibodies were used for the immunohistochemical analysis of HNE distribution in the brain.


Animal models: Defined hemorrhagic traumatic shock as well as sepsis (separate experiments) were performed as described elsewhere (6,7), on baboons (Papio Ursinus) weighing about 20 kg, sedated by ketamine hydrochloride and afterwards anesthesia was maintained with pentobarbital. Incomplete brain ischaemia was evoked in urethane anesthetized Wistar rats (approx. 450 g) by temporary occlusion of the carotids for 120 min. followed by 10 min. reperfusion (8).

Human samples: Brains tissue of people died from septic encephalopaty caused by different verified or not verified bacteria species.

Monoclonal antibodies against HNE and immunohistochemical analysis of the brain samples. Monoclonal antibodies for detection of HNE-modified proteins were obtained from the culture medium of the clone derived from a fusion of Sp2-Ag8 myeloma cells with B-cells of a BALBc mouse immunized with HNE modified keyhole limpet hemocyanine. The antibody is specific for the HNE-histidine epitope in HNE-protein (peptide) conjugates. For the immunohistochemical detection of the HNE-adducts the immunoperoxidase technique was used, with secondary rabbit-anti-mouse antibodies (Dako, Denmark), applied on 5µ sections of the formalin-fixed subserial paraffin embedded consecutive sections of the brain, as described before (6,7).

Results

Immunohistochemical analysis of the distribution of the lipid peroxidation product 4-hydroxynonenal (HNE) in the brain of baboons exposed to experimental hemorrhagic traumatic shock or sepsis showed that systemic oxidative stress in blood (blood vessels content) and the thereby generated HNE affect the blood-brain barrier and the regulation of cerebral blood flow determine secondary brain damage. In septic encephalopaty of the humans HNE affected, on the some way, the blood-brain barrier and subarachnoidal space like in animal experimental model. Similarly, HNE was determined during ischaemia in the brain blood vessels of rats exposed to ischaemic injury of the brain. After reperfusion, HNE disappeared from the blood vessels but remained in neurons and in glial cells.


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Summary of immunohistochemical findings of the HNE distribution in the brain of baboons exposed to hemorrhagic traumatic shock or sepsis, human brains with septic encephalopaty and rat brain exposed to ischaemia/reperfusion injury is presented in Table 1.

Hemorrhagic traumatic shock in baboons

Septic encephalopaty in baboons

Septic encephalopaty in humans

Ischaemia/reperfusion injury in rat brain

Main morphological findings according to the HNE- immuno-

positivity

HNE affects the blood-brain barrier and determine

secondary brain damage. The strongest HNE

positivity is in midbrain neurons, and neurons of

border zones

HNE affects the blood-brain barrier and

determine secondary brain damage. The strongest HNE positivity is in

astrocytes within white matter and in midbrain

neurons

HNE affects the blood-brain barrier and

determine secondary brain damage. The strongest HNE positivity is in

astrocytes within white matter and in midbrain

neurons

During ischaemia HNE affects the brain blood vessels, neurons and

glial cells. After reperfusion, HNE disappears from the blood vessels

but remains (less strong immunopositivity) in neurons and

in glial cells

Table 1. Immunohistochemical positivity to HNE-protein adducts determined in the brain of baboons exposed to hemorrhagic traumatic shock or defined sepsis (according to the references 6, 7 and 8), in human brains with septic encephalopaty and in rat brain exposed to ischaemia/reperfusion injury.

Fig 1. Immunohistochemistry of HNE (positivity is stained brown) in the brain tissue of rats exposed to I/R injury (x 400).

Apparently, anatomical and histological distribution of the ROS-derived products (like HNE), as a causative mechanism of the secondary brain damage, depends on the blood supply and pathophysiological features of the model applied. These findings are in agreement also with the immunohistochemical findings of HNE in the blood vessels of rats exposed to brain ischaemia/reperfusion (I/R) injury (Figure 1).

HNE was determined during ischaemia in the neurons (in the center of the upper photo) and in blood vessels (upper photo center and lower right corner) of rats exposed to brain ischaemia. After reperfusion (lower photo), HNE disappeared from the blood vessels (center and right side), but remained in some surrounding neurons and glial cells.


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Discussion

Due to findings on different HNE distribution in the particular regions of the brain we assume that intensity of the oxidative stress in the neurons of the septic baboons and baboons exposed to the hemorrhagic traumatic shock was influenced by two major parameters: 1) anatomy of the brain and 2) differential blood supply (6,7). The finding of the strong HNE-positivity of the astrocytes at the blood-brain barrier further indicates a humoral origin of the HNE adducts and an altered function of the blood-brain barrier under the pathological conditions studied (6,7). In agreement with that were findings on HNE positivity defined experimental sepsis as well as clinical findings on the brains of patients who died from septic encephalopathy. Furthermore, finding of the disappearance of HNE in the brain blood vessels of the rats after reperfusion in ischaemia-reperfusion model, although HNE was detected in the neurons after reperfusion raises a possibility that the disappearance of HNE after reperfusion might be accompanied by its further spread in the blood and brain tissue (8). However, this possibility should be further evaluated. Nevertheless, it appears certain that HNE is one of major mediators of oxidative stress in different pathological condition in brain acting like ”second toxic messenger” of lipid peroxidation in blood, brain-blood barrier and in brain tissue.

Literature Esterbauer H, Weger W. Über die Wirkungen von Aldehyden auf gesunde und maligne Zellen; Synthese von homologen 4-Hydroxy-2-alkenalen. Chemical Monthly 1967: 98: 1884-91

Zollner H, Schaur R J, Esterbauer H. Biological activities of 4-hydroxyalkenals. In: Oxidative stress. (Ed) Sies H, Academic Press, London-San Diego 1991: 337-69

Esterbauer H, Schaur R J, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal malonaldehyde and related aldehydes. Free Radic Biol Med 1991: 11: 81-128

Smith KJ, Kapoor R, Felts PA. Demyelination: The role of reactive oxygen and nitrogen species. Brain Pathology 1999:9:69-92

Waeg G, Dimsity G, Esterbauer H: Monoclonal antibodies for detection of 4-hydroxynonenal modified proteins. Free. Rad 1996: 25 149-59

Schlag G, Zarkovic K, Redl H, Zarkovic N, Waeg G. Brain damage secondary to hamorrhagic traumatic shock in baboons. In Shock, Sepsis and Organ Failure, G. Schlag, H. Redl, D.L. Traber (eds), Springer-Verlag, Heidelberg 1997: 3-17

Zarkovic K, Zarkovic N, Schlag G, Redl H, Waeg G. histological aspect of sepsis-induced brain changes in a baboon model. In Shock, Sepsis and Organ Failure, G. Schlag, H. Redl, D.L. Traber (eds), Springer-Verlag, Heidelberg 1997:146-60

Zarkovic N, Zarkovic K, Schaur RJ, Stolc S, Schlag G, Redl H, Waeg G, Borovic S, Loncaric I, Juric G, Hlavka V. 4-Hydroxynonenal as a second messenger of free radicals and growth modifying factor. Life Sci, 1999, 65: 1901-1904


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Age- and Sex-Dependent Oxidant and Antioxidant Status of CBA Mice

Sandra Sobocanec ([email protected]), Tanja Marotti, Visnja Sverko, Tihomir Balog and Ivo Hrsak

Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka c. 54, 10000 Zagreb, Croatia tel.+38514561172; fax+38514561010

Despite of numerous reports of lipid peroxidation and antioxidant enzyme status in mice, the results are still conflicting because most of the studies were not comprehensive concerning strain, sex and age of mice. In our study of oxidative and antioxidative status, we defined the dynamics regarding to age and sex in male and female CBA mice. By definition of these parameters, we can more reliable investigate the effect of various therapeutic agents on these features. We defined the dynamics of oxidant and antioxidant status by determining thiobarbituric acid-reactive substances (TBARS) content, superoxide dismutase (SOD) and catalase (CAT) activity in liver of 1, 4, 10 and 18 months old male and female CBA mice. TBARS content was assayed according to Ohkawa’s method (Ohkawa et al. 1979.); SOD activity was assayed according to Flohe’s method (Flohe et al. 1984.) and CAT activity according to Ou’s method (Ou et al. 1995.). TBARS content significantly increased with age (P<0.01) with no significant difference between sexes of the same age group. SOD activity varied with age in both sexes, significantly decreasing in young male mice (4 months old, P<0.01) and remaining unchanged in female mice until 18 months of age, when it markedly decreased (p<0.01). The changes observed in CAT activity were age- and sex-related; while in males CAT activity continuously increased with age (P<0.01), in females it decreased at 4 and increased at 18 months of age (P<0.01). Thus, it seems that changes in oxidative status are rather age- than sex-related, whereas antioxidative enzyme activities are age- and sex-related.

Ohkawa H. Ohishi N. Yagi K. (1979) Analytical Biochemistry 95; 351-358

Flohe L. Ötting F. (1984) Methods in Enzymology 105; 93-126

Ou P. Wolff S.P. (1996) Journal of Biochemical and Biophysical Methods 31; 59-67


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Age Changes of Lipid Peroxidation in Liver and Thymus of CBA and AKR Mice

Visnja Sverko, Tihomir Balog, Sandra Sobocanec, Mirjana Gavella* and Tanja Marotti

Division of Molecular Medicine, Laboratory of Biological Response Modifiers, Rudjer Boskovic Institute Bijenicka cesta 54, 10000 Zagreb, Croatia

* Vuk Vrhovac Institute for Diabetes, Endocrinology and Metabolic Diseases, Dugi dol 4A, Zagreb, Croatia

Oxidative damage occurring in the process of aging or degenerative diseases seems to be at least partly a consequence of the imbalance between formation and removal of free radicals. Contradictory results regarding the age related behavior of the oxidant process in the literature have been described. Both increases and decreases of lipid peroxidation have been found as specific changes in developing or aging processes, in various species, organs, tissues or sex.

The aim of this study was to determine the age- and sex- associated differences in oxidant status in the liver and thymus from CBA and AKR mice. Since AKR mice spontaneously develops leukemia about 4-6 months after birth our attention was also focused to oxidant status of AKR mice related to ontogeny of thymic lymphoma and age in both sexes.

Male and female CBA and AKR mice aged 3, 6 or 12 months were used. Oxidant status of CBA and AKR mice was estimated according to the presence of thiobarbituric acid reactive substances (TBARs) in the liver and thymus.

The results showed that TBARs increased significantly with age in the liver of both sexes in CBA mice. Also, significant increases in liver TBARs concentration of 12-month-old tumor-free AKR mice of both sexes was associated with aging too. On the contrary, thymus TBARs concentration was significantly increased only in males at 6 months of age, and it persisted to 12 months. TBARs concentration in female thymuses was unchanged during aging in both strains. A different pattern of TBARs concentration was seen in the liver (12-month-old) and thymus (6- and 12-month-old) of AKR mice with developed thymomas. In both organs and sexes TBARs concentration was significantly lower than in tumor-free AKR mice.

In summary, oxidant status in CBA mice is age-related in the liver in both sexes, while in thymus is age-related in male mice only. However, it seems that oxidant status in AKR mice are more closely linked with appearance of thymic lymphoma than being tissue- or sex- associated.


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Glutathione, the Most Important Ubiquitous Endogenous Antioxidant

Wonisch W.*, Halwachs, G.*, Grill D.#, Schaur R.J.^

* Department of Laboratory Medicine, University of Graz, Auenbruggerplatz 15, A-8036 Graz, Austria # Institute of Plant Physiology, University of Graz, Schubertstrasse 43, A-8010 Graz, Austria

^Institute of Molecular Biology, Biochemistry and Microbiology, University of Graz, Schubertstrasse 1, A-8010 Graz, Austria

The ubiquitous tripeptide glutathione (L-gamma-glutamyl-L-cysteinylglycine) occurs in virtually all cells as in animal cells and also in most plants and bacteria in concentrations ranging from 0.5 - 10mM. It is possibly the most abundant redox scavenging molecule in cells and its content is unexpectedly complex dependent on the carbon source supplied and varies as a function of age e.g. stationary phase cells showing a high glutathione content whereas logarithmic growth is associated with low levels of glutathione. It is the most prevalent intracellular thiol, which functions directly or indirectly in many important biological phenomena.

It participates in the reduction of disulfides and other molecules, and conjugates with compounds of exogenous and endogenous origin. Furthermore, its function is necessary for the synthesis of proteins and deoxyribonucleotide precursors of DNA and in the protection of cells against the effects of free radicals as well as of reactive oxygen intermediates. These are formed in metabolism e.g. photoscavenging of hydrogen peroxide produced during photosynthesis and in detoxifying hydrogen peroxide produced outside of the chloroplasts. Moreover, it is involved in the inactivation of a number of drugs, in metabolic processing of certain endogenous compounds, such as estrogens, prostaglandins and leukotrienes and is a coenzyme for several enzymes.

The cellular turnover of glutathione is associated with its transport out of the cell, in its reduced form GSH. Glutathione thus appears to be a storage- and transport-form of cysteine. The generality of GSH-transport implies this process being a protective mechanism for cell membranes, since there exists no extracellular mechanism for reduction of GSSG. Therefore, GSH must be continuously supplied from the cell.

GSH is of major importance in the reduction of peroxides through the action catalyzed by selenium-containing GSH-peroxidase and proteins with GSH-S-transferase activity. In this respect, GSH participates in the destruction of free radicals and peroxides, which are formed in metabolism under physiological conditions (e.g. phagocytosis) as well as after irradiation, under increased oxygen tension and through the administration of certain drugs.

The intracellular GSH may be particularly critical in cells in direct contact with the outside world, such as lung an intestine in mammals and chloroplasts, vacuoles and cytoplasm in plants. The synthesis of glutathione in plants occurs in the chloroplast and is thought to occur by the series of reactions as utilized in mammals called the ã-glutamyl cycle.

The BSO (buthionine sulfoximine) model - a model for endogenously produced oxidative stress:

Amino acid sulfoximines are specific inhibitors of gamma-glutamylcysteine synthetase. The most commonly employed is buthionine sulfoximine (BSO). BSO decreases the GSH level 80 to 90% in comparison to control cells. It inhibits the resynthesis of GSH, which replaces the GSH that is normally exported from the cell.

Deficiency of GSH (BSO model) demonstrates the need for cellular protection from endogenous reactive oxygen species (ROS). It is of particular importance for mitochondria, where the remaining glutathione turns over very slowly. Mitochondria do not synthesize GSH, but effectively transport it from the cytosol. Due to the lack of catalase mitochondria are particular dependent on the protection of GSH and GSH peroxidase. Long term treatment with BSO in adult mice leads to mitochondria swelling and vacuolization. The response of rodents upon BSO treatment is a substantially increase of plasma triglycerides and cholesterol levels, whereas tissue ascorbate levels are decreased. Recently, a very similar response was reported in the unicellular eukaryote Saccharomyces cerevisiae. Treatment with 4-hydroxy-nonenal, a GSH depleting hydroxyaldehyde originating from the peroxidation of ù-6-polyunsaturated fatty acids, leads to the fragmentation of the vacuole and to an accumulation of lipids in the cytosol.

Cerebral cortex, lung, liver and proximal tubule are mainly damaged due to BSO treatment, whereas heart and stomach are not affected.

On the other hand all these damages could be largely prevented by treatment with GSH monoesters and by ascorbate. Thus, ascorbate spares GSH and there exist apparently overlapping functions between ascorbate and GSH as well as between GSH and catalase.


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In the case of severe GSH deficiency in man, gamma-glutamylcysteine is overproduced and converted to 5-oxoproline, which accumulates, leading to acidosis, which is a secondary effect of GSH deficiency. As a consequence of GSH deficiency brain dysfunctions, hemolysis, peripheral neuropathy, myopathy and aminoaciduria occur.

A better understanding of basic mechanisms will be able with genetically manipulated yeast strains e.g. glutathione-deficient mutants (gsh1 and gsh2) with stable low GSH levels. These mutants for example are still viable although they have a slower growth rate and show a defect in sporulation. gsh1 mutants are absolutely dependent upon exogenous GSH for growth in minimal medium and display a petite phenotype, being unable to grow on non-fermentable carbon sources such as glycerol. On the other hand, gsh2 mutants can grow in unsupplemented minimal media due to the availability of gamma-glutamylcysteine.

Depletion of cellular GSH is in clinical trials as a combination anticancer therapy. It might make tumor cells more susceptible to irradiation and certain chemotherapeutic agents. Additionally it might be useful in treating infections due to antibiotic resistant microorganisms and parasites with respect to the hypersensitivity of these cells in comparison to the host. On the other side an increase of glutathione would be helpful against oxidative stress and in the protection against xenobiotics. Increased synthesis of GSH might be achieved by increasing the supply of substrates to the two synthetases. Due to the fact, that the first step in GSH synthesis is controlled by feedback inhibition, supply of gamma-glutamylcysteine is preferably associated with high GSH levels because the feedback-regulated step is bypassed. In S. cerevisiae GSH can also be used as a source of nitrogen and sulfur under starvation conditions.

Antioxidative effects of GSH:

ROS have been widely implicated in the pathology of numerous diseases such as atherosclerosis, ARDS, Parkinson´s, ischemia-reperfusion injury, rheumatoid arthritis and cancer.

In diseases, associated with oxidative damage low thiol levels were reported e.g. in the epithelial lining fluid and peripheral blood mononuclear cells (PBMC) of HIV infected patients. Low PBMC levels of GSH were also found in chronically infected hepatitis C patients and people suffering from type II diabetes are reported to have decreased GSH and increased GSSG levels in whole blood.

One of the best explanations for the protective effect of ascorbate and GSH in atherosclerosis comes from Willis (1953). It was found that in cholesterol fed scorbutic guinea pigs atherosclerosis was inhibited in those given ascorbate. Ascorbate and GSH decrease the formation of oxidized LDL, which is the form implicated in the development of atherosclerosis.

ROS and their peroxy products are detoxified by the GSH redox-cycle. Glutathione acts nonenzymatically as a radical scavenger with the redox-active sulfhydryl group with oxidants. GSH peroxidase (and non-Se peroxidase) catalyzes the destruction of hydrogen peroxide and hydroperoxides.

Furthermore, GSH plays an important role in the metabolism of xenobiotics, undergoes conjugation reactions with electrophilic agents either spontaneously or mediated by GSH-S-transferase leading to the detoxification of substances, which are potential mutagens or carcinogens. In the biliary tree and intestine or in the kidney the GSH conjugate is then attacked by GAMMA-glutamyltranspeptidase, which removes the GAMMA-glutamyl moiety, leaving a cysteinyl-glycine conjugate. This conjugate is cleaved by a dipeptidase, resulting in a cysteinyl conjugate. Finally, a mercapturic acid is formed by the acetylation of cysteine.

Among different yeast strains Hansenula mrakii was shown to be most resistant against linoleic acid hydroperoxide by the induction of glutathione peroxidase, which reduces the hydroperoxide moiety to the alcohol moiety.

HNE-treatment caused an initial depletion of the glutathione content in Saccharomyces cerevisiae cells, macrophages, monocytes and skin fibroblasts, which was followed by an increase, up to a level approximately twice as high in comparison to control cells, due to de novo synthesis. Moreover, HNE was reported as a good substrate for cytosolic glutathione transferases from rat.

Furthermore, glutathione deficient mutant strains were reported to be hypersensitive against hydrogen peroxide, suggesting that intracellular glutathione plays an important role in the adaptive response to oxidative damage in S. cerevisiae. Endogenously produced hydrogen peroxide is reduced by GSH in the presence of selenium-dependent GSH peroxidase. As a consequence GSH is oxidized to GSSG, which in turn is rapidly reduced back to GSH by GSSG reductase at the expense of NADPH, thereby forming a closed system (redox cycle).

Due to the important role as an antioxidant the maintenance of a high reduced-oxidized ratio inside the cell is provided by the enzyme glutathione-reductase. Mutations in the GSH-reductase gene are associated with an accumulation of an excess of oxidized GSH and a hypersensitivity to oxidants.


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References: Glutathione Meister A. & Anderson M.E. Ann. Rev. Biochem. 1983; 52:711-760

Selective modification of glutathione metabolism Meister A. Science 1983; 220:472-477

Glutathione metabolism and its role in hepatotoxicity DeLeve L. D. and Kaplowitz N. Pharmac. Ther. 1991; 52:287-305

Glutathione and glutathione delivery compounds Anderson M.E. Antioxidants in disease mechanisms and therapy Eds. Sies H. Advances in Pharmacology 1997; 38:65-78 Academic Press

Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Esterbauer H.; Schaur R.J.; Zollner H. Free Rad. Biol. Med. 1991; 11:81-128

Increased stress parameter synthesis in the yeast Saccharomyces cerevisiae after treatment with 4-hydroxy-2-nonenal Wonisch W.; Hayn M.; Schaur R.J.; Tatzber F.; Kranner I.; Grill D.; Winkler R.; Bilinski T.; Kohlwein S.D. and Esterbauer H. FEBS Letters 1997; 405:11-15

Oxidative stress responses of the yeast Saccharomyces cerevisiae Jamieson D.J. Yeast 1998; 14:1511-1527


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Effects of Stobadine on Immunohistochemical Findings of HNE-Protein Adducts in Rat Brain Cells In Vitro

Iva Loncaric*, Kamelija Zarkovic*, Svorad Stolc**, Georg Waeg*** and Neven Zarkovic****

* Division of Neuropathology, Medical Faculty, KBC Zagreb, Kispaticeva 12, HR-1000 Zagreb, Croatia ** Institute of Experimental Pharmacology, Slovak Academy of Science, Bratislava, Slovakia

*** Institute of Molecular Biology, Biochemistry & Microbiology, Karl-Franzen's University, Graz, Austria **** Rudjer Boskovic Institute, Division of Molecular Medicine, Zagreb, Croatia

Introduction

Generation of oxygen radicals and the process of lipid peroxidation came in focus of attention in many fields including trauma and stroke of central nervous system (CNS) (1). CNS is particularly sensitive to oxygen radical damage because of the high levels of polyunsaturated lipids in neuronal cell membranes and the unique and sophisticated function these membranes have serving in neuronal signal transduction.

Traumatic injury involves physical disruption of vascular and neuronal tissue while ischaemic injury entails the interruption of blood flow. Subsequent to the primary injury series of events is initiated which leads to secondary tissue damage, and rapidly, traumatic injury evolves into an ischaemic insult. Aldehyde 4-hydroxynonenal (HNE) is a lipid peroxidation product generated during pathophysiological conditions based on the production of reactive oxygen species (ROS) (2). It is considered to be a causative factor of secondary tissue damage.

Pharmacological development and possible use of compounds that inhibit lipid peroxidation or scavenge oxygen radicals can be promising to promote survival and neurological recovery after CNS injury, especially if these antioxidants are used as prevention or very early after onset of injury. Pyridoindole Stobadine is novel compound with antioxidant and scavenging properties (3). Stobadine was shown to scavenge hydroxyl, peroxyl and alkoxyl radicals, to quench singlet oxygen, to repair oxidized aminoacids and to preserve oxidation of SH groups by one electron donation. These effects originated from its ability to form stable nitrogen-centered radical on indole nitrogen. Consequently, it was able to diminish lipid peroxidation and protein impairment under oxidative stress. Since the biochemical interactions and possible influence of Stobadine on HNE induced oxidative damage are unknown, the aim of this study was to analyze whether Stobadine interacts chemically with HNE and does it protect rat brain cells in vitro against HNE induced damage considering time of its administration (before or after the onset of oxidative damage induced with HNE).


Dot-blot analysis

Dot-blot analysis was performed on human albumin treated with HNE. Stobadine was added to the reaction mixture before or after HNE administration. Samples were applied on two nitro-cellulose membranes in three different concentrations (original sample, 10 fold, and 100 fold dilutions). Primary antibody against HNE-protein conjugate was added only to one membrane while the other was control (5). Detection of antibodies was conducted by measuring peroxidase activity of enzyme-marked tertiary antibody (PAP-mouse, Chemicon, USA) applying DAB staining. Peroxidase activity of sample enzymes was blocked (with 1,5% H2O2, 0.1% Na-azid, 2%BSA) before adding secondary antibody (rabbit-anti-mouse, Dako, Denmark).

Suspension of rat brain cells

Four-months-old male Wistar rats were anesthetized with chloral hydrate (300 mg/kg). Brain was removed under sterile conditions and cut into two hemispheres. One of them was fixed in 10% formalin. Brain-cell suspension was prepared from the other hemisphere by mechanical dispersion in cold RPMI-1640 medium with 20% FCS. The method was based on Messer's protocol, which was adjusted according to requirements of this experiment (4).

In vitro treatment with HNE and Stobadine

Cell suspensions were treated with 50 mM HNE for 30 minutes. Stobadine (10 mM) was added to the suspensions 30 minutes before or after HNE administration. After incubation, cells were centrifuged at 1100 rpm/min, resuspended in RPMI-1640 medium, centrifuged again and than the pellet was fixed 10% formalin in Eppendorf vial.

Immunocitochemistry on HNE-protein conjugates

Brain-cell pellets were dehydrated and paraffin embedded in Eppendorf vials. After hardening, paraffin was removed from vials and embedded again in standard paraffin blocks. Immunocitochemistry was performed on two 5 mm consecutive sections. For the immunohistochemical detection of the HNE-protein adducts, with the monoclonal


30

antibody for the HNE-histidine epitope, the immunoperoxidase technique was used (PAP mouse, Chemicon, USA) with secondary rabbit-anti-mouse antibodies (Dako, Denmark) (5, 6).

Results

The dot-blot analysis showed no visible difference in quantity of formed HNE-albumin conjugates when Stobadine was added to reaction mixture ( Fig.1).

Figure 1. Dot-blot analysis of albumin-HNE binding in the presence of Stobadine applied before or after HNE. Dilutions of the samples (in duplicates, from top to the bottom): original sample, 10-fold, and 100-fold dilutions. Control, in vitro untreated, rat brain cells were HNE negative. Cells exposed to 50 µM HNE in vitro showed high HNE positivity. Treatment with 10mM Stobadine diminished HNE positivity when Stobadine was administered before HNE. If Stobadine was administered after HNE there was no visible protective effect on cells. (Fig. 2).

Fig. 2 Immunocitochemistry of HNE in rat brain cells after in vitro incubation with HNE and Stobadine.

Discussion

Since Stobadine did not influence binding of HNE to albumin, as shown with dot-blot analysis, we can assume that that mechanism of antioxidant defense of Stobadine does not include direct physical and/or chemical interactions with HNE and preventing binding of HNE to proteins.

In contrary, in environment that include live cells in vitro, Stobadine shows protective effect against HNE induced oxidative stress. It probably protects cells acting as a scavenger for ROS formed during oxidative stress induced in cells after HNE treatment and possibly also by enchancing cell endogenous antioxidant potential which will be further studied.

Literature Hall ED, Braughler JM: Central nervous system trauma and stroke II. Free Rad Biol Med 1989; 6: 303-313.

Esterbauer H, Schaur RJ, Zollner H: Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Rad Biol Med 1991; 11: 81-128.

Horakova L, Stolc S: Antioxidant and pharmacodinamic effect of pyridoindole Stobadine. Gen Pharmacol 1998; 30: 627-38.

Messer A: The maintenance and identification of mouse cerebellar granule cells in monolayer culture. Brain Res 1997; 130:1-12.

Waeg G, Dimsity G, Esterbauer H: Monoclonal antibodies for detection of 4-hydroxynonenal modified proteins. Free Rad Biol and Med 1996; 25: 149-159.

Zarkovic K, Zarkovic N, Schlag G, Redl H, Waeg G: Brain damage secondary to hemorrhagic shock in baboons. Schock, Sepsis and Organ Failure, Springer- Verlag, Heilderberg 1997, 146-160.


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Oxygen Derived Free Radicals and Anesthesia

B. Mazul-Sunko*, I. Loncaric**, S. Borovic***, M. Peric*, M. Novkoski*, A. Krizmanic*, A. Gvozdenovic* and N. Zarkovic***

* Department of Anesthesiology and Intensive Care Medicine, University Hospital Sestre Milosrdnice, Zagreb, Croatia ** Department of Neuropathology, Medical Faculty & Clinical Center Zagreb, Croatia

*** Department of Molecular Medicine Rudjer Boskovic Institute, Zagreb, Croatia

Traditional concept is that anesthesiologist is a physician trained to give general anesthesia, while more appropriate opinions consider task of anesthesiologists as maintaining homeostasis during stress which can be either controlled event during elective surgical procedure, or an uncontrolled one in polytrauma or haemorrhagic shock.

In this context, the biology of free radicals is relevant in two ways:

1. Oxidative stress is considered important in pathophysiology of numerous clinical situations encountered in surgery and intensive care. It is involved in pathobiology of reperfusion injury, acute respiratory distress syndrome or myocardial depression in sepsis. Decreasing free radicals production can have very practical consequences: for example cardiac dysfunction in patients who were operated using extracorporal circulation pump is proved to be partially caused by free radicals. Therefore, patients with off-pump by-pass have less cardiac complications (1). High doses of methilprednisolone in acute spinal injury is proved an effective treatment because it decreases lipid peroxidation in nervous tissue and attenuates neurologic deficit. Metilene blue, a NO antagonist, improves hypotension in pediatric patients with septic shock, but does not influence mortality.

2. Anesthetics themselves might have pro- and anti- inflammatory effects.

If anesthetics themselves can modulate oxidative stress, is topic of several current clinical and experimental investigations, that also attracted us to perform preliminary study in vitro.

Anesthetics as generators of free radicals

Metabolism of inhalation anesthetic halothane has been extensively investigated because halothane induced hepatitis is a rare, but potentially fatal event. It seems that reactive intermediates of halothane oxidative metabolism, which cause trifluoracetilation of tissue proteins, are responsible for the halothane hepatotoxicity (2). Another anesthetic, which can generate free radicals, is ketamine and therefore some investigators do not recommend its use in animal experimental research on free radicals (3).

Anesthetics as potential free radical scavengers

A possibility that anesthetics can reduce free radical caused tissue damage is very attractive. It means that by giving anesthetics homeostasis derangement caused by free radical generation can be modulated. In this way reperfusion injury during cardiac, vascular or neurovascular surgery could by theoretically prevented. Therefore, numerous investigations analyze this possibility. But the results are conflicting.

Two intravenous anesthetics, propofol and thiopental, are found to be free radical scavengers in majority of in vitro and in vivo models of oxidative stress. Thiopental has been extensively used by neurosurgical anesthesiologists because it has numerous beneficial effects in acute brain injury, among others free radical scavenging properties. It is an important effect because it has been proved that free radicals scavengers like desmethyl tirilazid improve neurologic deficit in an animal model of ishaemic brain injury (4). Thiopental and methohexital decrease hydroxyl radical production, lipid peroxidation a hypoxic cell death in human NT-2 neurons (5). Controlled clinical trials, however, proved its therapeutic effects only in certain subgroups of patients, for example young patients with severe head injury and during clamping of carotid artery during endartherectomy.

At the moment, the most promising anesthetic acting as free radical scavenger seems to be propofol. It was found that combination of alpha tocopherol and propofol completely abolish free radical generation in neuronal cells (6). The effect of propofol on oxidative stress in platelets of surgical patients was studied and it was found that propofol attenuates oxidative stress by decreasing TBARS and increasing glutation (7). Comparative study in the presence of erythrocyte membranes proved that propofol is superior to midazolam regarding antioxidant activity and suggested it could be a better sedative agent in critically ill patients who are treated for illnesses based on oxidative stress (8). It was also found that propofol attenuates mechanic and metabolic changes in rat heart induced by exogenously applied hydrogen peroxide (9). On the other hand, no protective effect on stunned heart was found. (10). Another study found that propofol has free radicals scavenging properties, but in doses 10 times higher than are necessary for general anesthesia (11).


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Inhalational anesthetics halothane and isoflurane have antioxidant properties as well, but it seems that that have no direct antioxidant effects, but are mediated through iron ions (12).

We have done preliminary study on effects of anesthetics on hydrogen peroxide formation in an in vitro model that uses hydrogen peroxidase as a marker of hydrogen peroxide generation. Four anesthetics were used: propofol, thiopental, fentanyl and midazolam. Thiopental significantly attenuated peroxide/peroxidase activity, and propofol completely abolished it. Midazolam and fentanyl were ineffective.

Although the effects of propofol were found in dose ten folds above the dose applied in clinical conditions during general anesthesia, our results are consistent with results of other in vitro models of the effects of the particular anesthetic on oxidative stress and implies its applicability in further free radicals research in anesthesiology.

Literature 1. Matata BM et al. Off pump by-pass graft operation significantly reduces oxidative stress and inflammation. Ann. Thoracic Surgery 69:785-791, 2000

2. Baden JM et al in Anesthesia. Third Edition :149-153. Edited by Miller RD. et al.

3. Reinke LA. Free radical formation during ketamine anesthesia in rats – a cautionary note. Free Radical Biology & Medicine 24:1002-1006,1998

4. Feng YZ et al Desmethyl tirilazad improves neurologic function after hypoxic ishemic brain injury in piglets. Critical Care Medicine 28:1431-1438, 2000

5. Almaas R. et al. Effect of barbiturates on hydroxyl radicals, lipid peroxidation, and hypoxic cell death in human NT-2 neurons. Anesthesiology.92.764-774, 2000

6. Boland A. Propofol protects cultured brain cells from ion –induced death: comparison with trolox. European Journal of Pharmacology.404.21-27, 2000

7. De la Cruz et al. The effect of propofol on oxidative stress in platelets from surgical patients. Anesthesia & Analgesia. 89:1050-1055, 1999

10. Tsuchiya M. et al. Propofol versus midazolam regarding their antioxidant activities. American Journal of Respiratory & Critical Care Medicine.163:26-31,2001

11. Kokita N et al. Propofol attenuates hydrogen peroxide-induced mechanical and metabolic derangaments in the isolated rat heart. Anesthesiology. 84: 117-127, 1996

12. Ross S. Et al. A comparison of the effects of fentanyl and propofol on left ventricular contractility during myocardial stunning. Acta Anaesthesiologica Scandinavica. 42:23-31,1998

13. Green TR and al. Specificity and properties of propofol as an antioxidant free radical scavenger. Toxicology & Applied Pharmacology. 129:163-169,1994

14. Kudo M. Absence of direct antioxidant effects from volatile anesthetics in primary neuronal-glial cultures. Anesthesiology. 94:303-312,2001


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Carcinogenic and Mutagenic Effects of Reactive Oxygen Species and Lipid Peroxidation Products

R. Jörg Schaur

Institute of Molecular Biology, Biochemistry and Microbiology, University of Graz, Graz, Austria

The human adult consumes about 660 g oxygen per day. 90 - 95 % of that oxygen are converted by mitochondrial respiration to harmless water, but the remaining 5 - 10 % of oxygen undergo univalent and divalent reduction yielding reactive oxygen species (ROS) such as superoxide radicals, hydrogen peroxide and hydroxyl radicals. Extrapolated to the entire lifespan the human body produces the huge amount of 800 - 1700 kg of ROS. These ROS cause the endogenous oxidative degradation of membrane lipids by lipid peroxidation, which results in the formation of a very complex mixture of lipid hydroperoxides, chain cleavage products, and polymeric material.

Experimental animal studies and biochemical investigations lend support to the hypothesis, that lipid peroxidation products, either produced endogenously or ingested with food, represent a health risk. The oral toxicity of oxidized lipids is unexpectedly low, probably thanks to the various antioxidative systems of the body. But chronic uptake of large amounts of such materials increases tumor frequency and the incidence of atherosclerosis in animals. Spalding et al. found that the lipid peroxidation product malon-dialdehyde is carcinogenic in mice and rats. 4-Hydroxynonenal, an aldehydic chain-cleavage product resulting from omega-6 polyunsaturated fatty acids, such as linoleic and arachidonic acid, induces several genotoxic effects in hepatocytes and lymphocytes. The concentrations of the aldehyde needed to produce these effects are in the range expected to occur in vivo. E.g. Eckl et al. reported that 0.1 µM are sufficient to induce increased levels of sister-chromatid exchange in primary cultures of rat hepatocytes.

Further studies are needed to clarify the pathophysiological significance of these findings.


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HNE and TGF-ββββ1 Expression in the Cancerogenesis of the Colon

Fiorella Biasi§, #, Daniella Zanetti#, Juan C. Cutrin #, Barbara Zingaro#, Elena Chiarpotto# and Giuseppe Poli#

§ CNR Centro di Immunogenetica e Oncologia Sperimentale e # Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale S.Luigi, Regione Gonzole 10,

10043 Orbassano (Torino), Italy

An increasing number of reports underlines the frequent association of fibrosclerotic diseases of lung, liver, arterial wall, etc. with accumulation of oxidatively modified lipids and proteins, aldehydic end-products.

A cause-effect relationship between cellular oxidative damage and increased fibrogenesis has been proposed on the basis of either prevention or quenching of the fibrotic process by experimental treatment with antioxidants.

With some peculiarities in the different organs, fibrosclerosis essentially results from the interaction of macrophages and extracellular matrix-producing cells.

The cross-talk is mediated by fibrogenic cytokines, among which the most important appears to be the transforming growth factor β1 (TGF-β1).

It is well established by many studies on lipid peroxidation in biological samples that a great variety of aldehydes are produced following the decomposition of membrane lipid hydroperoxides (Poli, et al., 1985; Esterbauer et al., 1991). In particular, the most represented aldehyde of the hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE) shows a wide spectrum of biochemical and likely biological effects.

Previous studies reported that treatment of different types of human and murine macrophages with HNE, induce consistently both m-RNA expression and synthesis of TGFβ1 (Chiarpotto et al., 1997). Thus, lipid peroxidation can indeed be involved in the dysregulation of macrophage function occurring in organ diseases with sclerotic evolution.

Since increased steady state levels of HNE (at micromolar concentrations) have been demonstrated in the cirrhotic liver and in the human oxidatively modified low density lipoproteins associated to atherosclerosis, the up-regulation of macrophage TGFβ1 by the aldehyde could contribute to such and analogous diseases characterized by fibrosclerosis (Muller et al., 1996, Poli and Parola, 1997, Leonarduzzi et al., 1997).

Susceptibility to lipid peroxidation during malignant transformation is known to be inversely related to the degree of dedifferentiation. TGFβ1 has been shown to inhibit proliferation of a wide range of normal parenchyma cell types, while in many cases, cancer cells display resistance to its growth-inhibitory effect.

The reduced susceptibility to the lipid peroxidation and the decrease of the expression of TGFβ1 in tumors can be correlated. To this purpose, we examined the possible correlation between the expression and synthesis of TGFβ1 and its receptors, and the reduced susceptibility to peroxidative reactions in fifteen adult patients affected by colon adenocarcinoma at different stages of dedifferentiation.

As expected, in bioptic samples of patients bearing colon adenocarcinoma the extent of lipid peroxidation was decreased as compared to the normal counterpart of tissue in relation to the malignancy progression. The different indices of redox balance were monitored in terms of tissue malonaldheyde (MDA) production and fluorescent adducts between HNE/MDA and proteins: of note, HNE appeared to be a more sensitive marker, in fact HNE-protein adducts were found to be significantly reduced in all T2 and T3 tumors (TNM classification)

Correspondingly, the same T2 and T3 colon cancers, almost all G2 differentiation degree, revealed a dramatic low content of TGFβ than that detected in surrounding normal colon (the evaluation of TGFβ was performed on biopsies with Western blot analyses).

The actual decreased expression of TGFβ seems to be fundamental in the escape mechanism of tumor cells from tissue host defenses.

A favorable condition for neoplastic progression of the tumor might be through the decrease of the extent of oxidative reactions with a consequent diminished formation of the antiproliferative lipid peroxidation aldeydic-end products in tumor area (Schauenstein et al 1977), among them the HNE, that is able to contribute to the modulation of expression and synthesis of the fibrogenic cytokine; a depressed generation of such reactive molecules, like that occurring during neoplastic transformation, may be followed by a net decrease in TGF-β1 availability.

To evaluate if the TGFβ1 receptor system is implicated in the tumor progression process TGFβ1-deficiency dependent, the expression of type I (RI) and type II (RII) receptors was evaluated in tissue sections embedded with avidine-biotine system in area adjacent and distal from tumor site. The immunostaining evidenced an impairment of TGFβ1 receptor


35

system in the tumors; in particular the down-regulation was usually greater for TGF RI than for RII, however, not all individuals bearing cancers show the same impairment in the tumor mass; so, it was not possible to correlate this alteration with the tumor progression.

According to several studies, at least with regard to epithelial cells, from which colon carcinoma originates, TGFβ1 undoubtedly exerts a growth inhibitory effect, through an activating signal on both RI and RII (Wrana et al., 1992). Therefore, the alteration of receptor system may be crucial for the inhibitory effect of TGFβ1 on cell proliferation and transformation. However, the properties of these receptors, in particular type I, have not yet been fully characterized, and a differential role has been suggested for RI and RII proteins in the pleyotropic effects of the cytokine (Chen et al., 1993; Miettinen, et al., 1994).

We performed in our laboratory an immunocytochemical analysis for TGFβ1 receptor system in a human colon cancer cell line, CaCo-2 cells.

The semiquantitative analyses showed low levels of both TGFβ1RI and TGFβ1RII receptors: two thirds of the randomly examined cancer cells lack the type I receptor, while about two thirds of them still possess type II receptor. Of note, if it is actually necessary for TGFβ1-dependent growth inhibition that receptors I and II are fully expressed and acting together, the observed pro-apoptotic effect of this cytokine on CaCo-2 cells would not be possible. However, treatment of CaCo-2 cells with an appropriate concentration of TGFβ1 (5 ng/ml) for 48 h led to a marked induction of programmed death, as detectable both by nick end labeling (TUNEL) analyzed with confocal microscopy and by DAPI staining.

Furthermore the co-treatment of CaCo-2 cell line with TGFβ1 and 1 µM HNE showed an increased number of apoptotic bodies. Thus, the still present nuclear fragmentation in the CaCo-2 cells, notwithstanding the down regulation of TGFβ1 receptors, and its amplification due to the addition of HNE, indicates that TGFβ1 can signal even in the presence of a marked reduction of specific receptors.

In conclusion, the escape of the human colon cancer cells from TGFβ1-mediated growth inhibition does not appear as strictly dependent upon a down-regulation of TGFβ1 receptors, which is inconsistent and unrelated to cancer development, but in particular upon a constantly low concentration of this cytokine in the tumor mass. The associated levels of lipid peroxidation aldehydes, much lower than in control tissue, likely represent a lower stimulus for TGFβ1 production in the neoplastic area, thus a favorable condition for neoplastic progression.

References Chen, R.H., Ebner, R., Drynck, R. Inactivation of the type II receptor reveals two receptor pathways for the diverse TGF-beta activities. Science, 260:1344-8 ;1993.

Chiarpotto, E., Scavazza, A., Leonarduzzi, G., Camandola, S., Biasi, F., Mello-Teggia, P., Garavoglia, M., Robecchi, A., Roncari, A., Poli, G. Oxidative damage and transforming growth factor β1 expression in pretumoral and tumoral lesions of human intestine. Free Radic. Biol. Med., 22:889-894;1997.

Esterbauer, H., Schaur, R.J., Zollner, H. Chemistry and Biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med., 11:81-128,1991.

Leonarduzzi, G., Scavazza, A., Biasi, F., Chiarpotto, E., Camandola, S., Vogel, S., Dargel, R., Poli, G. The lipid peroxidation end product 4-hydroxy-2,3-nonenal up-regulates transforming growth factor beta1 expression in the macrophage lineage: a link between oxidative injury and fibrosclerosis. FASEB J., 11:851-7;1997.

Miettinen, P.J., Ebner, R., Lopez A.R., Derynck, R. TGF beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors. J Cell Biol., 127:2021-36;1994.

Muller, K., Hardwick, S.J., Marchant C.E., Law, N.S., Waeg, G., Esterbauer, H., Carpentar, K.L., Mitchinson, M.J. Cytotoxic and chemotactic potencies of several aldehydic components of oxidized low density lipoprotein for human monocyte-macrophages. FEBS Lett., 388:165-8;1996.

Poli, G., Dianzani, M.U., Cheesemann, T.F., Slater, T.F., Esterbauer, H. Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions. Biochem. J., 227:629-638;1985

Poli,G., Parola, M. Oxidative damage and fibrogenesis. Free Radic Biol Med., 22:287-305;1997.

Schauestein, E., Esterbauer, H., Zollner, H. Aldehydes in biological systems: their natural occurrence and biological activities. Pion Ltd. London, 1997.

Wrana J, Attisano L, Wieser R., Ventura, F., Massagué, J. Mechanism of the activation of the TGFb receptor. Nature 1994;370:341-7.


36

Some Aspects of Systemic Oxidative Stress in Surgery of Human Colon Carcinoma

Sabolovic S1 ([email protected]), Stipancic I1, Zarkovic N2, Romic Z3, Mayer LJ4, Martinac P1, Tatzber F.5, Zarkovic K. 6, Loncaric I6, Vukovic T2, Borovic S2

1 Department of Surgery, Clinical Hospital Dubrava, Zagreb, Croatia 2 Div. of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia

3 Central Biochemical Laboratory, Clinical Hospital Dubrava, Zagreb, Croatia 4 Department of Medical Biochemistry, Faculty of Pharmacology and Biochemistry, Zagreb, Croatia

5 Institute of Nuclear Medicine, Vienna, Austria 6 Institute of Neuropathology, Medical Faculty, KBC Zagreb, Zagreb, Croatia

Introduction

Colon and rectal cancers are the leading causes of morbidity and mortality from all gastrointestinal malignancy, worldwide. In the United States, colorectal cancer is the third most common cause of cancer related mortality (1). The cause, as well as biological properties, of colorectal cancer remain elusive, although numerous studies revealed multifactorial involvement. In recent years, the possible ethiological and prognostic role of reactive oxygen species (ROS) are intensively investigated (2).

An overproduction of reactive oxygen species, biochemicaly defined as oxidative stress, is involved in a large variety of processes and diseases, including carcinogenesis and aging (3). The development of cancer can be stimulated by oxygen free radicals, which do not cause cell death. The end products of lipid peroxidation, such as aldehydes and epoxides, have been shown to be mutagenic (4) and to be able to favor neoplastic transformation through unstable binding to DNA. Once transformed, cancer cells become more resistant to lipid peroxidation (and oxidative damage) than their normal counterparts (5).

In this study we measured some markers of oxidative stress in patients with colorectal adenocarcinoma before and after radical surgery. We, also evaluated a possible relationship between oxidative stress induced lipid peroxidation and colorectal carcinoma by HNE-immunohistochemistry.

Patients and Methods

Blood was taken from patients with colorectal cancer who undergoing radical colorectal resection. Six patients were included in the study with different tumor stage: from T2 to T4.

Twenty healthy adults provided normal reference values for plasma indices of oxidative stress, and normal tissue distant from the tumor lesions was used for comparative immunohistochemistry analysis.

Blood samples were obtained preoperatively and 24, 72 hours postoperatively and on the seventh day after surgery. Heparinized whole blood samples were used for sera preparation. For the analysis of SOD activity blood samples were used for lysate preparation. The samples were stored at -40oC. During colorectal radical resection tissue samples were taken - normal tissue distant from the tumor lesions well as the tumor tissue.

In tissue samples we analyzed distribution of HNE-protein conjugates by immunohistochemisty (PAP method, DAB staining with hematoxyllin contrast staining) applying genuine monoclonal antibodies.

Levels of endogenous peroxides in the sera samples were detected by enzyme spectrophotometric assay (POX, eliTec). Superoxide dismutase was determined from lysates of erythrocytes by RANSOD kit (Randox, Crumlin, UK). The enzyme activities were related to the hemoglobin content of the haemolysates.

Results

In patients with colorectal cancer we noticed significant increase of peroxide activity (POX) in respect to mean control values. On the third day after surgery POX level significant decreased in comparison to preoperative level and, also, to the control. Such initial decrease was followed by returning to normal values on seventh day after operation, although that still represent decrease level in order to value we measured before operation (fig 1).


37

)

Figure 1. Peroxide activity level in patients with colorectal carcinoma before and after surgery in comparison to control values. *according to the Student-s t-test, significantly different due to control level (p< 0,05)

** according to the Student-s t-test, significantly different due to preoperative level (p< 0,05

Figure 2. Preoperative and postoperative level of superoxide dismutse in lysates of erythrocytes in patients with colorectal carcinoma in comparison to control values. *according to the Student-s t-test, significantly different due to control level (p< 0,05)

Significant decrease in level of superoxide dismutase (SOD) in lysates of erythrocytes was observed in patients with colorectal carcinoma in comparison to mean control values (fig 2). Increase of SOD activity was noticed after surgery. Such pattern of dynamic change persisted on seventh day after surgery, when the SOD values became again below the level of SOD activity in healthy controls.

Immunohistochemistry revealed that the tumors (malignant cells) were not associated with formation of HNE-protein conjugates. Blood vessels were also negative, although erythrocytes inside vessels showed HNE-positivity. The only HNE-immunopositivity which was noticed in tumor tissue was due to infiltration of the tumors with inflammatory cells which were HNE-positive (fig 3).

Figure 3. Immunohistochemistry of 4-hydroxynonenal-protein conjugates in carcinoma and in adjacent normal colon tissue . In normal colon tissue (right on both photos) distant from the tumor lesion (upper photo left), and in tumor itself HNE-immunopositivity (brown) was observed only in inflammatory cells (lower photo right side). Occasionally, submucosal blood vessels were positive. In other area of normal colon tissue HNE-immunopositivity was not observed. Upper photo - 50x original magnification; lower photo - the same part higher (200x) original magnification.

POX

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38

Discussion

Observed preoperative higher level of hydrogen peroxide in carcinoma patients indicate systemic oxidative stress related to cancer development as systemic disorder. Moreover, significant reduction of peroxide levels which occurred on the third day after surgery seems to be a result of tumor removal and consequential reduction in the metabolism of reactive oxygen species (systemic oxidative stress). The decrease of peroxides measured in that point could be partly dependent upon increased activity of antioxidant systems due to tumor removal. Finally, serum peroxides were equal to the control values also on the seventh day suggesting that possible normalization of ROS activity might have occurred.

We assume that lower preoperative SOD level in carcinoma patients as result of its negative regulation and possible suppression of complete antioxidant system. Increase of SOD after surgery might be due to intraoperative and postoperative administration of blood or erythrocytes that could result in higher level of SOD in respect to preoperative level. However, a week after surgery SOD was lower than in healthy controls support a concept of cancer as systemic disorder manifested also as persistent oxidative stress.

In malignant tissue we did not observe abundant presence of HNE-protein conjugates. That might be related to a reduction in membrane content of polyunsaturated fatty acids. Thus, by keeping lipid peroxidation processes at very low levels, tumor cells could protect themselves from the cytostatic effects of lipid peroxidation-derived molecules (4,6). However, oxidative stress resulting in HNE production will be further present in cancer tissue at least due to the inflammatory response, as noticed, which is apparently one of the crucial mechanisms of the tumor/host relationship.

References 1. Greenlee GT, Murray T, Boleden S et al. Cancer statistics 2000. CA Cancer J Clin 2000;50:7-34.

2. Emerit I. Reactive oxygen species, chromosome mutation and cancer: possible role of clastogenic factors in carcinogenesis. Free Radic Biol Med 1994;16:99-109.

3 Knight JA. Diseases related to oxygen-derived free radicals. Ann Clin Lab Sci 1995;25:111-121.

4. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 1991;11:81-128.

5. Dianzani MU. Lipid peroxidation and cancer. Crit Rev Oncol/Hematol 1993;15:125-147.

6. Chiarpotto E, Scavazza A, Leonarduzzi G, Camandola S, Biasi F, Teggia PM, Garavoglia M, Robecchi A, Roncari A, Poli G. Oxidative damage and transforming growth factor β1 expression in pretumoral and tumoral lesions of human intestine. Free Radic Biol Med 1997; 22:889-894.


39

Ischemia-Reperfusion of Walker 256 Carcinoma

Suzana Borovic1 ([email protected]), Iva Loncaric2, Senka Sabolovic3, Andreas Meinitzer4, Tea Vukovic1, Kamelija Zarkovic2, Renate Wildburger5, Manfred Tillian6, Georg Waeg6,

Pero Martinac3, Igor Stipancic3 and Neven Zarkovic1 1 Department of Molecular Medicine, "Rudjer Boskovic" Institute, Zagreb, Croatia

2 Department of Neuropathology, Clinical Hospital Center "Zagreb", Zagreb, Croatia 3 Department of Surgery, Clinical Hospital “Dubrava”, Zagreb, Croatia

4 Department of Laboratory Medicine, Blocklabor 1, University of Graz, Graz, Austria 5 University Clinic of Traumatology, University of Graz, Graz, Austria

6 Institute of Molecular Biology, Biochemistry and Microbiology, Karl-Franzens University of Graz, Graz, Austria

Introduction

Oxygen free radicals, or reactive oxygen species (ROS) are produced and released in organism usually in metabolic processes. In some cases, such as shock, sepsis, trauma, hemorrhage, hypoxia, ischemia-reperfusion, ROS are produced in excess resulting in a condition termed oxidative stress. One of the most harmful ROS is the hydroxyl radical - HO· due to its very high reactivity and instability. ROS can attack macromolecules - DNA, proteins, sugars and lipids, causing their damage and consecutively be cytotoxic. Reaction of ROS with unsaturated fatty acids cause lipid peroxidation, having as end-products different aldehydes. One of such aldehydes is 4-hydroxynonenal (HNE), generated during pathophysiological processes based on the production of ROS and considered as a causative factor of secondary tissue damage (1,2,3,4).

ROS also play well-known role in carcinogenesis, and ischemia-reperfusion presents a model for potentiating ROS production (1,2). The purpose of this work was to investigate HO· formation and extent of lipid peroxidation in ischemia-reperfusion of Walker 256 carcinoma and compare it with possible morphological changes determined by immunohistochemsitry of HNE-protein adducts in the tumor tissue.

Materials and Methods

Walker carcinoma tumor cells (107 live cells) were injected i.m. in the hind limb of male Wistar rats. The experiment was done after 6 days when the tumor was visible. Rats were divided in three groups: 1) control group (anaesthetized only); 2) operated (laparatomy); 3) operated rats treated by ischemia-reperfusion. Ischemia-reperfusion (I/R) of the tumor was performed by clamping the ipsilateral iliac artery. Rats were treated i.v. with acetylsalicylic acid (ASA), 20 mg/kg of body weight and anaesthetized with chloralhydrate (300 mg/kg). Plasma as well as tissue samples were collected after 90 minutes of ischemia and 30 minutes of reperfusion. Plasma was kept frozen at –20 oC while tissue was stored in formalin.

For determination of dihydroxybenzoic acids (DHBAs) samples were extracted with hydrochloric acid and diethylether with 3,4-DHBA as internal standard. Separation was performed on a 150 x 4,6 Waters Spherisorb ODS2 3 µm column using mobile phase of 7,48 mM sodium citrate/acetic acid pH 4,6 with 3 % of methanol. Detection was done with an electrochemical detector (ESA Detector Coulochem 2, with 5040 analytical cell model) set on 400 mV and flow of 0,6 ml/min. Concentrations of all compounds extracted from plasma were recalculated according to the internal standards and calibration curve.

As 2,5-DHBA can be produced also by an enzymatic pathway, only 2,3-DHBA can be applied in HO. production quantification (5).

The HPLC method for 2,3-DHBA determination is in use for measuring HO. production in different tissues (6-7). We have used this method to investigate Walker 256 carcinoma tissue response to ischemia-reperfusion. 2,3-DHBA concentrations were recalculated according to the salicylic acid concentrations measured in the plasma and expressed as percentage of salicylic acid.

Significance was calculated according to the Student’s t-test and a value of p<0,05 was considered as significant.

Tissue was used for immunohistochemical determination of HNE-modified proteins with monoclonal antibodies raised against HNE-histidine conjugates by the PAP method (4).


40

Results and Discussion

DHBAs separation results obtained from rat plasma are presented in Figure 1. On the chromatogram we can see two metabolites of salicylic acid: 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA); and 3,4-DHBA which was used as internal standard.

Results presented in Figure 2 show an increase in 2,3-DHBA production when tumor was treated by I/R (p<0.05).

Also, we wanted to see the extent of lipid peroxidation after ischemia-reperfusion by immunohistochemical staining against HNE, one of the end-products of lipid peroxidation. Results obtained were presented on Figure 3; We have shown by staining tissue sections with monoclonal antibodies against HNE-histidine conjugate that there was no increase, but rather a decrease, in lipid peroxidation caused by ischemia-reperfusion of the tumor.

Figure 2. 2,3-DHBA concentrations in the plasma expressed as an percentage of salicylic acid. Increase in 2,3-DHBA production was measured when Walker 256 tumor was treated by ischemia-reperfusion. Three groups of rats were presented: 1) control, non-treated; 2) operated (laparatomy); 3) operated with ischemia-reperfusion.

Figure 1. HPLC chromatogram of rat plasma showing two metabolites of salicylic acid: 2,3-dihydroxybenzoic acid (2,3-DHBA), 2,5-dihydroxybenzoic acid (2,5-DHBA); and 3,4-DHBA that was used as internal standard.


41

So, it seems that even if increased HO. radical production in ischemia-reperfusion injury was present, there was no consequent increase in lipid peroxidation in the tissue or at least no formation of HNE-protein adducts, which might have been even "washed away" during a blood reflow, as was noticed before for the incomplete brain ischemia/reperfusion (4). Decreased content of polyunsaturated fatty acids in cancer cell membrane and probably increased antioxidants defense in the tissue might also contribute to this phenomenon (8).

Acknowledgement

The authors would like to thank IFCC for providing Professional Scientific Exchange Scholarship to Ms. Suzana Borovic and to the Austrian Nationalbank that made part of this work possible. The support of the Croatian Ministry of Science and Technology is kindly acknowledged.

References 1. Braughler JM, Hall ED. Central nervous system trauma and stroke: I. Biochemical considerations for oxygen radical formation and lipid peroxidation. Free Rad Biol Med, 1989, 6:289-301.

2. Hall ED, Braughler JM. Central nervous system trauma and stroke: II. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. Free Rad Biol Med, 1989; 6:303-313.

3. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde and related aldehydes. Free Rad Biol Med, 1991, 11:81-128.

4. Zarkovic N, Zarkovic K, Schaur RJ, Stolc S, Schlag G, Redl H, Waeg G, Borovic S, Loncaric I, Juric G, Hlavka V. 4-hydroxynonenal as a second messenger of free radicals and growth modifying factor. Life Sci, 2000, 65:1901-1904.

5. Ingelman-Sundberg M, Kaur H, Terelius Y, Persson JO, Halliwell B. Hydroxylation of salicylate by microsomal fractions and cytochrome P-450: lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted. Biochem J, 1991, 276:753-757.

6. Choudray C, Talla M, Martin S, Fatome M, Favier A. High-performance liquid chromatography – electrochemical determination of salicylate hydroxylation products as an in vivo marker of oxidative stress. Anal Biochem, 1995; 227:101-111.

7. Doppenberg EMR, Rice MR, Di XO, Young HF, Woodward JJ, Bullock R. Increased free radical production due to subdural hematoma in the rat – effect of increased inspired oxygen fraction. J Neurotrauma, 1998; 15:337-347.

8. Slater TF. Lipid peroxidation and cell division in normal and tumor tissues. In: Eicosanoids, lipid peroxidation and cancer. Nigam SK, Mc Brien DCH, Slater TF (eds). Springer-Verlag, 1988, 137-142.

Figure 3. Immunohistochemical staining of tumor sections by anti-HNE monoclonal antibodies. Brown staining indicate presence of HNE. Left photo presents control tumor while right presents ischemia-reperfusion treated tumor. There is no increase in HNE-positivity after ischemia-reperfusion.


42

Cytostatic Effects of Michael-Adducts of αααα,ββββ- unsaturated Aldehydes on Model Tumors of Rats and Mice

H. Manfred Tillian and Silvia Piendl

Institute of Molecular Biology, Biochemistry and Microbiology, Karl-Franzen’s University, Graz Austria

Introduction:

α,β- unsaturated aldehydes show toxicity for cells, especially tumor cells. These effects were initially shown when applied peritumorally on solid tumors. Intraperitoneal treatment, however, did not show any effects upon either solid or ascitic growing tumors. The agent is rapidly inactivated mainly by conjugation with glutathione. To avoid these unwanted effects another property was used: α,β- unsaturated aldehydes react reversibly with another thiol-compound, cysteine, forming Michael addition products (1:1- adduct) or a thiazolidine carboxylic acid (1:2- adduct) respectively. Adducts release the aldehyde again, slowly and gradually. Adducts can therefore act as a storage form for these tumor inhibitory agents and increase their effectiveness.

Although growing very fast, Walker-256-carcinosarcoma is a more realistic tumor model in order to improve the study of cytostatic effects. In this investigation the cells are grown in solid form in the liver of rats. The agent is administered subcutaneously using a coated tablet to keep it at a constant level in plasma.

Materials & Methods

Agents:

Cysteine-1:1-hydroxypentenal-adduct, molecular weight: 221, melting point: 145C. This adduct is only used for treatment of EAT in mice.

Cysteine-2:1-crotonal adduct (CAS 4170-30-3), molecular weight: 294,4, melting point: 168- 171C; synthetized in our lab by condensation of cysteine with crotonal.

Treatment:

Ehrlich Ascites Tumor, EAT, (strain Heidelberg Lettrè) in Swiss mice (ascitic and solid) doubling time: 48 hours; lifespan after inoculation: about 3 weeks and 6-8 weeks respectively. 5 doses are administered intraperitoneally 72, 96,120, 144 and 168 hours after implantation of 4-5 x 105 cells/ 10g b.wt.

Walker 256- Carcinosarcoma in Sprague Dawley rats (infiltration of 4-5 x 105 cells into the liver via vena portae to allow taking of the tumor in solid form); lifespan: 4 to 5 days. Application of tablets containing the adduct subcutaneously following infiltration of tumor cells simultaneously (i.e. tablets and cells were administered at the same time) or the tumor was allowed to grow unaffected for 24 hours or 48 hours respectively. Each treated group was accompanied by a sham-operated control.

Tablet: Retard-implantation tablet, administered subcutaneously into the neck of the animal; liberation of the agent within 8 hours

Results:

EAT in mice: Strongest therapeutic effects are seen using crotonal (2:1)- and hydroxypentenal (1:1)- adducts.

Cysteine (1:1)- hydroxypentenal- adduct: Controls treated animals

Total died before 90th day Survived 90th day

10 29 16 13

Extension of survival period for treated animals, which died before 90th day, is, dose-dependent, between 0 and 89%.


43

Cysteine (2:1)-crotonal- adduct: Controls treated animals

Total died before 90th day Survived 90th day

10 10 7 3

Extension of survival period for treated animals, which died before 90th day is 5%.

Wa-256 in rats: tumor developed taking of tumor avoided

Inoculation and treatment simultaneously : 0 20

Delay of treatment for 24 hours: 0 20

Delay of treatment for 48 hours: 14 0

Discussion:

Early experiments in vitro and in vivo, show the antitumor activity of a,b- unsaturated aldehydes, mainly 4-hydroxy-pentenal(HPE) administered peritumorally on EAT. Applied intraperitoneally, however, the effects remained poor, because the agents are reabsorbed and inactivated in the blood. Systemic administration therefore seemed impossible.

As shown above treatment of EAT bearing animals with Michael adducts is possible; but there was no success in treating animals bearing the tumor in solid form. Since Michael-adducts exist in solid form it is possible to store that agents in depot-tablets. Using tablets the release of agents is adjustable to time as well as to dosage.

The next goal of our study was to prove that systemic administration of a,b- unsaturated aldehydes is possible by subcutaneous implantation of tablets, thus improving the penetration of the drug into the tumor. As an improved model, Walker-256-cells, implanted into the liver, are in use. It is shown, that the concentration of the agent in tumor-cells is 2 to 44 times higher, depending on the circulatory status of the tumor-bearing animal, than in the surrounding liver tissue. These promising findings lead to a treatment of solid Walker-256-carcinosarcoma, inoculated into the liver, using a systemic route from a subcutaneous depot via the blood to the tumor. It is shown, that taking of the tumor is avoidable depending on date of implantation of the depot.

References Esterbauer, H., Ertl, A., Scholz, H., The Reaction of Cysteine with α,β- Unsaturated Aldehydes. Tetrahedron, 32, 285 (1976)

Tillian, HM., Schauenstein, E., Ertl, A., Esterbauer, H., Therapeutic Effects of Cysteine Adducts of α,β- Unsaturated Aldehydes on Ehrlich Ascites Tumor of Mice. Europ.J.Cancer, 12, 989-993 (1976)

Tillian, HM., Schauenstein, E., Esterbauer, H., Therapeutic Effects of Cysteine Adducts of α,β- Unsaturated Aldehydes on Ehrlich Ascites Tumor of Mice II. Europ.J.Cancer, 14, 553-536 (1978)

Schauenstein, E., Esterbauer, H., Submolecular Biology and Cancer. CIBA Foundation Series, 67, 225 (1979)

Piendl, S. Diploma Thesis. Institute of Pharmaceutical Chemistry, Karl Franzens University of Graz (1988)

Tillian, HM., Wintersteiger, R., Gübitz, G., Systemic Application of Cysteine- 2.1- Adduct for Pharmacokinetic Studies of Tumor Models in Rats and Mice. Drug Res.,46, 640-642 (1996)


44

Cancer and Granulocytes

R. Jörg Schaur

Institute of Molecular Biology, Biochemistry and Microbiology, University of Graz, Graz, Austria

Abstract Neutrophil polymorphonuclear granulocytes, the largest population of leukocytes, act as phagocytes within the human immune system. They posses a high potential to destroy tumor cells by oxidative and non-oxidative mechanisms. These mechanisms, with special emphasis on the myeloperoxidase-hydrogen peroxide-halide system, are described in this contribution as well as the defense strategies, which tumor cells employ to escape cytolysis.

1. Introduction An early model of the interaction of neutrophil granulocytes with tumor cells

2. The oxidative and non-oxidative armory of phagocytes The role of myeloperoxidase (MPO) The oxidative and chlorinating potential of hypochlorite

3. Neutrophils can destroy both cancer cells and normal cells Not all cell types are killed by identical mechanisms

4. Among the non-oxidative mechanisms are cationic proteins and hydrolases

5. The cytolytic mechanism depends in most cases on the respiratory burst The MPO-hydrogen peroxide-halide system has a cytotoxic effect on several mammalian tumor cells MPO may behave as a device to amplify lysis The cytotoxicity activating stimuli, PMA and IgG-coated targets, follow different transductional pathways to

trigger the effector cell lytic systems There are exceptions from the dependence on the respiratory burst

6. How can target cells protect themselves against the attack of neutrophils ? Not all potential target cells are killed by neutrophils Target cells have to be "primed" by antibodies Target cells can metabolize oxidants such as hydrogen peroxide Target cells posses antioxidative enzymes However, antioxidative enzymes cannot always prevent from oxidative attack HOCl traps can act as down-modulators of the cytotoxic process In cancer patients the migratory activity of neutrophils can be reduced

7. Neutrophils may interfere with other cytolytic mechanisms of the immune system

8. Therapeutic approaches exploiting the tumoricidal activity of MPO

9. Systemic effects: The role of phagocytes in cancer cachexia

10. A special case: carcinogenesis and inflammation


45

Up-Regulated NEP and APN Activity and Down-Regulated LPO Content in Patients with Pheochromocytomas

Tihomir Balog1, Tanja Marotti1, Visnja Sverko1, Miljenko Marotti2 and Ivan Krolo2 1Div. of Molecular Medicine, Institute “Rudjer Boskovic”, Zagreb, Croatia

2Dept. of Radiology, University Hospital “Sestre Milosrdnice”, Zagreb, Croatia

Patients with adrenal medullary pheochromocytoma, but not patients with non-functional adrenal tumors, have elevated content of enkephalins. APN (aminopeptidase N, CD13) and NEP (enkephalinase, CALLA, CD10) peptidases present on the cells from various tissues, including neutrophils, are main enzymes responsible for degradation of enkephalins in vivo. We have compared the APN and NEP activity of neutrophils from patients with adrenal gland tumors (ten feochromocytomas and twelve non-functional adrenal gland tumors), human tumors of different origin (n=9) and 15 healthy donors. In the same patient we also measured lipid peroxidation (LPO) in the plasma. The diagnosis of each patient was confirmed with endocrinology analysis computed tomography (CT) and/or postoperative histology analysis. NEP activity of neutrophils from patients with pheochromocytomas was up regulated (p=0.001) compared to NEP activity of neutrophils from patients with non-functional adenomas, other tumors or healthy persons. Besides only NEP activity, (but not APN activity or LPO content) in pheochromocytoma patients significantly and consistently decreased 15 days after surgery as compared to level before surgery (n=5). APN activity in pheochromocytoma patients was also up regulated (p=0.01) compared to APN activity in patients with non-functional adenomas, other tumors or healthy persons. Contrary to that LPO was elevated in patients with non functional adenomas and other tumors (p<0.05) and unchanged in pheochromocytomas as compared to healthy persons. Thus, NEP activity might be an additional discriminating parameter in diagnosis of pheochromocytomas.


46

Methods for Measurement of Oxidative Stress and Lipid Peroxidation

Franz Tatzber*, Ulrike Resch* and Willibald Wonisch**

* University of Vienna, Inst. of Nuclear Medicine, Währinger Gürtel 18/20, A-1090 Vienna, Austria ** University of Graz, Medical Clinic, Auenbruggerplatz 1, A-8036 Graz

One of the most common definitions for oxidative stress (OS) is the excess of oxidants compared to antioxidants. Therefore in OS situations the capacity of antioxidants to neutralize the oxidizing effects of prooxidative substances (i.e. radicals, peroxides, etc) is exhausted and lipids and cell membranes are “left to the mercy of oxygen”.

Lipid peroxidation (LPO), especially LDL oxidation is probably the most intensely investigated phenomenon of OS. LDL oxidizes in three consecutive phases. During the lag phase antioxidants are consumed and only minimal production of oxidation products can be observed. After the entire consumption of chain breaking antioxidants oxidation products like free radicals, peroxides and aldehydes are produced during the propagation phase. The generation of oxidation products shows the characteristics of a chain reaction. During the last phase, the degradation or decomposition phase, this chain reaction breaks down due to the lack of oxidizable substances. LDL and its carrier protein Apolipoprotein B 100 (ApoB 100) are degraded to smaller particles.

Methods for measurement of OS and/or LPO events include direct methods, which detect free radicals, conjugated dienes or other LPO products, and indirect methods, which measure antioxidants or LPO events by degradation of e.g. oxidation sensitive dyes.

A. Direct methods

A1. Electron Spin Resonance (ESR) This method allows the detection of unpaired electrons characteristic for free radicals. The fact, that free radicals can be determined quantitatively, is the main advantage of ESR. Its main disadvantage is that it requires expensive equipment and is therefore restricted to specialized laboratories. Of all methods ESR has the best future perspectives as a very reliable method for measurement of OS effects.

A2. Measurement of conjugated dienes: This method is based on the fact that during lipid peroxidation polyunsaturated fatty acids (PUFAs) develop double bonds which are separated by a single bond (= - =), commonly addressed as conjugated dienes. This diene formation can be measured online at 234 nm. Its main advantage is that LPO can be followed online. Its main disadvantage is the fact that it requires complicated sample preparation including ultra centrifugation and therefore like method A1 it is restricted to specialized labs.

A3. Measurement of peroxides: Several sufficient methods for peroxide detection have been described. Among the most common there are luminometric and colorimetric detection of hydroperoxides and the detection of lipid hydroperoxides by a complexometric method. The main advantages of the methods are that they are relatively easy to perform. Their main disadvantages are that they can measure only parts of the spectrum of peroxides in a pH dependant manner.

A4. Measurement of thiobarbituric acid reactive substances (TBARS): TBARS are mainly aldehydes and the most important aldehyde in this measurement is malonic dialdehyde (MDA). The measurement of TBARS is a relatively simple colorimetric method. However, unless unspecific color reactions are separated by HPLC, it lacks of specificity and can only be used as a screening method. Separation by HPLC requires expensive equipment and restricts the method to specialized laboratories. However, together with A2 the measurement of TBARS contributed very much to the current knowledge of OS and LPO.

B. Indirect Methods

B1. Measurement of antioxidants: Several HPLC methods allow very precise measurements of secondary antioxidants. Unfortunately these methods require complicated sample preparation and are also restricted to specialized laboratories. On the other hand, colorimetric methods for detection of total antioxidant capacities are simple procedures, but usually they do not discriminate between vitamin antioxidants and substances with antioxidative properties, such as uric acid, albumin and bilirubine.

B2. Measurement of oxidizability of lipoproteins (LIPID-OX): LIPID-OX is a simplification of the conjugated diene method described in A2. It is based on the destruction of an oxygen sensitive fluorescence dye, which is extremely lipophilic. Because it does not require excessive sample preparation, this method is easier adaptable for the requirements of routine laboratories. However, the need of a fluorimeter is a limiting factor for wide spread application of this method.


47

B3. Measurement of antibodies to oxidized LDL (oLAb): It is a simple ELISA method, which is adaptable to almost any routine laboratory. Its disadvantage is the fact that by this method only the immune reaction to OS and LPO is determined.

B4. Measurement of oxidized LDL: Also a simple ELISA method, which can be used in almost any clinical laboratory. The disadvantage is that there is no exact definition for oxidized lipoproteins and therefore possible cross reactions to native lipoproteins cannot be excluded.

In conclusion, there is quite a number of reliable methods for measurement of OS and LPO. It can be stated that generally specificity and sensitivity are lost by increasing simplicity of the methods. Although the situation concerning reliable methods for OS and LPO has markedly improved during the last years, there is still a demand for methods, which are easily applicable in routine laboratories and reliable enough to produce valid results for the measurement of OS and LPO.


48

Oxidative Stress in Heart Diseases

Vrkic N.*, Nikolic-Heitzler N.*, Zarkovic N.**, Borovic S.**, T. Vukovic**, Tatzber F.*** and Topic E.*

* - Clinical Hospital "Sestre Milosrdnice", Zagreb, Croatia ** - "Rudjer Boskovic" Institute, Division of Molecular Medicine, Zagreb, Croatia

*** - Institute of Nuclear Medicine, Vienna, Austria

Cardiovascular diseases are associated with altered lipid metabolism, which is even considered as a major risk factor of atherosclerosis and consequential development of relative occlusion of blood vessels causing increased blood pressure and coronary diseases that might lead to acute myocardial infarction.

Altered lipid metabolism is based on increased intake of fat associated with oxidative modification of lipids, in particular low density lipoproteins (LDL) resulting in oxidative stress (OS). Aiming to evaluate if OS based on lipid peroxidation and production of oxidized LDL (oLDL) is progressively manifested or occurs mostly in the final onset of disease (acute cardiac disease) we analyzed parameters of lipid metabolism, amount of lipid peroxidation products (LPO) and antibody titer against oLDL in patients with angina pectoris (N=32), with acute myocardial infarction (N=36) and in normal healthy subjects (N=52).

Although total cholesterol levels and LDL-cholesterol were above normal values only in patients with angina pectoris (p<0.001) (Table 1), not in those with myocardial infarction (p>0.05, Table 2), other parameters of lipid metabolism were equally altered in patients with angina pectoris and with myocardial infarction. Hence, in both groups levels of HDL-cholesterol were significantly lower than in healthy subjects, while triglycerides were increased above normal (p<0.05).

Parameter

Healthy persons N=52

median (min-max)

Patients with angina pectoris N=32

median (min-max)

Mann-Whitney U-Test

p level

oLAb (mU)

583.5 (93.0-1177.0)

471.5 (72.0-1687.0)

0.475

LPO (µmol/L)

2.5 (0.7-5.6)

1.9 (0.8-4.7)

0.274

Cholesterol (mmol/L)

5.7 (3.00-8.85)

6.7 (4.0-12.4)

0.001

HDL-chol (mmol/L)

1.2 (0.59-2.26)

0.9 (0.5-1.7)

<0.001

LDL-chol (mmol/L)

3.6 (0.4-5.7)

4.6 (0.93-10.67)

<0.001

Triglicerids (mmol/L)

1.3 (0.53-5.50)

2.2 (1.0-7.2)

<0.001

Table 1. Comparison of lipid metabolism and lipid peroxidation between healthy persons and patients with angina pectoris.

Parameter

Healthy persons N=52

median (min-max)

Patients with acute myocardial infarction N=36

median (min-max)

Mann-Whitney U-Test

p level

oLAb (mU)

583,5 (93.0-1177.0)

282 (27-2114)

0.0014

LPO (µmol/L)

2,5 (0.7-5.6)

4,4 (1.50-11.1)

<0.001

Cholesterol (mmol/L)

5,7 (3.00-8.85)

5,9 (3.7-9.2)

0.491

HDL-chol (mmol/L)

1,2 (0.59-2.26)

1,0 (0.4-1.8)

<0.001

LDL-chol (mmol/L)

3,6 (0.4-5.7)

4,2 (1.49-7.36)

0.064

Triglicerids (mmol/L) 1.3 (0.53-5.50)

1,8 (0.7-4.1)

0.036

Table 2. Comparison of lipid metabolism and lipid peroxidation between healthy persons and patients with acute myocardial infarction.

However, OS-damaged lipids seem to occur only in patients with myocardial infarction. Namely, in these patients significant increase of LPO was detected (p<0.001) while anti-oLDL titer was decreased (p=0.0014), while these values


49

were normal in patients with angina pectoris (p>0.1). Thus, it could be concluded that altered lipid metabolism is in patients with angina pectoris manifested at least as much as in patients with myocardial infarction, while oxidative damage of lipids, in particular LDL develops mostly in myocardial infarction, not in patients with angina pectoris. Immune response to this, manifested by the production of antibodies against oLDL appears to play important role in acute onset of cardiac disease, while its interference with the OS in chronic cardiac patients is further studied intensively.

References Esterbauer H., Dieber-Rotheneder M., Waeg G., Striegl G., Jürgens G.: Biochemical, structural and functional properties of oxidized low density lipoprotein. Chem Res Toxicol, 3:77-92, 1990;

Salonen J.T., Ylä-Herttuala S., Yamamoto R., Butler S., Korpela H., Salonen R., Nyyssönen K., Palinski W., Witztum J.L.: Autoantibody against oxidized LDL and progression of carotid atherosclerosis. Lancet, 339:883-887, 1992;

Schumacher M., Eber B., Tatzber F., Kaufmann P., Esterbauer H., Klein W.: Oxidized LDL-autoantibodies in patients with coronary artery disease. Lancet, 340:123-127, 1992;

Vrkic N., Zarkovic N., Nikolic-Heitzler V., Topic E., Tatzber V., Vukelic N., Kalisnik T. Monitoring of oxidative stress markers in patient with acute myocardial infarction. Acta Clin Croat, 36: 85-88, 1997;

Moore K., Roberts J.L.: Measurement of lipid peroxidation. Free Rad Res, 25:659-671, 1998;

Letimäki T., Lehtinen S., Solakivi T., Nikkilä M., Jaakkola O.: Autoantibodies against oxidized low density lipoprotein in patients with angiographically verified coronary artery disease. Arterioscler Thromb Vasc Biol, 19:23-27, 1999;


50

Serum Peroxides In Patients With Cholelithiasis - Differences Between Laparotomic And Laparoscopic Surgery

Stipancic I.1 ([email protected]), Sabolovic S.1, Zarkovic N.2, Romic Z.3, Busic Z.1, Martinac P.1, Tatzber F., Loncaric I.5, Borovic S.2, Vukovic T.2

1 Department of Surgery, Clinical Hospital Dubrava, Zagreb, Croatia 2 Department of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia

3 Central Biochemical Laboratory, Clinical Hospital Dubrava, Zagreb, Croatia 4 Institute of Nuclear Medicine, Vienna, Austria

5 Institute of Neuropathology, Medical Faculty, KBC Zagreb, Zagreb, Croatia

Introduction

Trauma, whether accidental or surgical, induces local as well as systemic neuroendocrine, immune and metabolic response (1). The magnitude of this stress response reflects the severity of tissue trauma and surgery. Laparoscopic surgery has become the procedure of choice for treatment of symptomatic cholelithiasis as well as some other abdominal diseases due to it obvious clinical benefits: small lesion of the abdominal wall, less pain and faster recovery (2). However, little is known about mechanisms of the observed benefits, although it has been postulated that laparoscopic procedure is less traumatic, associated with less perioperative stress and, therefore, with diminished neuroendocrine-immune and metabolic response.

Several studies have analyzed the surgical stress and biologic response induced by laparoscopic cholecystectomy compared with an open approach (laparotomy), but although it seems that laparoscopic procedures cause less stress, there is no general agreement if it is so for different stress-parameters (3,4).

Surgical injury is associated with increased production of reactive oxygen species and utilization of antioxidant defense systems (5). Many experimental data are in support of a damaging role of excess of oxidative reactions, also termed oxidative stress, in systemic stress responses after surgical injury.

In this study some markers of oxidative stress and level of oxidative damage after laparoscopic and conventional open cholecystectomy have been compared in a randomized trial.

Patients and Methods

In a prospective study, some parameters of oxidative stress were measured in the blood of 43 patients underwent elective surgery. Because of symptomatic cholecystolithiasis twenty-two patients (group 1) underwent laparoscopic cholecystectomy and the data were compared with twenty-one patients (group 2) submitted to open cholecystectomy. Twenty healthy adults provided normal reference values for the sera indices of oxidative stress.

Blood samples were obtained preoperatively and 24, 72 hours postoperatively and on the seventh day after surgery. For the measurement of anti-oLDL titer of autoantibody EDTA plasma was immediately prepared and sucrose (60 mg/ml) was added as 10% to prevent LDL decomposition and further peroxidation. For others performed analysis heparinized whole blood samples were used for sera preparation. The samples were stored at -40oC.

Autoantibodies against oxidized LDL were determined by enzyme linked immunosorbent assay (oLAb, eliTec). Levels of endogenous peroxides were detected by enzymatic spectrophotometric assay (POX, eliTec).

Results

In both, laparoscopic and open groups, statistical significant decrease of anti-oLDL autoantibodies (oLAb) titer was seen preoperatively in comparison to mean control values (Fig. 1). Thus, the decrease of anti-oLDL titer in order to control values, persisted in both groups in all points we measured.

There was no statistical significant difference in oLAb titer between laparoscopic and open group in any point evaluated.


51

Figure 1. Preoperative and postoperative values of oxidized LDL antibody titer in laparoscopic and open cholecystectomy in comparison to control values

Although, oLAb values showed a continuos decreasing trend in laparoscopic group after operation that was significant differences if compared with preoperative values. Opposite to that, in group operated by open approach, oLAb values after operation in each point showed significant increase after surgery (Fig.1).

In both groups, there was no difference in preoperative values of peroxide activity (POX) in comparison to control values (Fig. 1). Postoperatively significant difference in POX values, in order to healthy controls was seen only on the seventh day after surgery in laparoscopic group. Such a difference was not noticed in group with open approach (laparatomy), hence, there was not significant differences at any point after surgery in comparison to the control values.

In laparoscopic group POX values decreased first day after operation in respect to preoperative values. That initial decrease of POX level was followed by an increase of POX values on seventh day after surgery, when POX values were higher than measured before surgery. The same pattern of dynamic change in POX level was noticed in group operated by open approach, although postoperative decrease was even more pronounced (Fig. 2).

Figure 2. Peroxide activity level in patients undergoing laparoscopic and open cholecystectomy in comparison to control values

Discussion

It is supposed that decreased titer of autoantibodies measured in both groups before surgery is a consequence of oxidative processes which appeared because of inflammatory disease associated with altered lipid metabolism (cholelithiasis and chronic cholecystitis). Hence, there might an excess of oLDL, which capture anti-oLDL antibodies. Afterwards, a higher titer of autoantibodies could be synthesized, as it was observed in group operated by open

oLAb

0

200

400

600

800

1000

1200

beforesurgery

I postopday

III postopday

VII postopday

A 4

50 LAP

CONTROL

OPEN

POX

0200400600800

10001200140016001800

beforesurgery

I postopday

III postopday

VIIpostop

day

A 4

50 LAPCONTROLOPEN


52

approach, because of intensity of surgical injury and consequential oxidative stress. It seems that enhanced production of autoantibodies did not occur in laparoscopic patients because less intensive surgical injury.

Decreased level of peroxide activity, as it was measured in both groups one day after surgery, can be consequence of antioxidative system activity due to the traumatic (surgical) injury (recruiting antioxidative defense of the body). Increased production of peroxides noticed in both groups afterwards, we assume can be result of feedback utilization of antioxidative parameters or as a consequence of enhanced oxidative processes which appeared after injury and decreased food intake that might affect both metabolism in general and in particular metabolism of the antioxidants and lipid peroxidation.

References 1. Weissman C. The metabolic response to stress: an overview and update. Anesthesiology 1990;73:308-27.

2. Schirmer BD, Edge SB, Dix J, Hyser MJ, Hanks JB, Jones RS. Laparoscopic cholecystectomy. Treatment of choice for symptomatic cholelithiasis. Ann Surg 1991; 213:665-77.

3. Targarona EM, Pons MJ, Balague C et al. Acute phase is the only significantly reduced component of the injury response after laparoscopic cholecystectomy. World J Surg 1996;20:528-34.

4. Karayiannakis AJ, Makri GG, Mantzioka A, Karousos D, Karatzas G. Systemic stress response after laparoscopic or open cholecystectomy: a randomized trial. Br J Surg 1997;84:467-71.

5. Poli G, Cutrin JC, Biasi F. Lipid peroxidation in the reperfusion injury of the liver. Free Rad Res 1998;28:547-551.


53

Oxidative Stress and its Dependence on Depth and Total Burn Surface Area

M Mujadzic*, T. Vukovic**, S. Borovic**, A. Cipak**, I. Loncaric***, F. Tatzber**** and N Zarkovic**

* - University Clinical Center Sarajevo, Sarajevo, Bosnia and Herzegovina ** - Rudjer Boskovic Institute, Division of Molecular Medicine, Zagreb, Croatia

*** - Division of Neuropathology, Medical Faculty, Clinical Center Zagreb, Zagreb, Croatia **** - Institute of Nuclear Medicine, Vienna, Austria

Introduction

It is assumed that the increase of lipid peroxides in the period after burn injury (as a part of "burn toxins") could cause damage of various organs by allowing leakage of different enzymes in to the blood, or significantly contribute to the toxicity of the sera and induce organ damage leading to ARDS, MOF and death as the final result.

Oxidative stress is basic mechanism of posttraumatic lipid peroxidation, as well as low density lipoprotein (LDL) peroxidation. Peroxidation of LDL cause chain reaction resulting in production of cytotoxic oxidatively modified "oLDL" which can be metabolized only by mononuclear cells with an inflammatory reaction as consequence, and even with synthesis induction of autoantibodies against oLDL (Virella G. et al., 1993; Borovic S. et al. 1995).

It appears that the oLDL titer is in direct correlation with the outcome of the disease and the course of recovery (Schumacher M. et al. 1995). Therefore would monitoring of intensity of oxidative stress be relevant to predict posttraumatic course of the illness (Stipancic I. and Zarkovic N. 1997).

In our study we have examined dependence of oxidative stress (by determing level of total capacity of antioxidants and a titer of autoantibodies against oLDL ) on a depth and burn surface what has not been done before, to improve the monitoring of convalescence and therefor morbidity and mortality of the patients.

Material and Methods

Patients

The study included 55 patients divided in 4 groups according to surface and depth of the burned tissue. Group I contained 12 patients superficially burned up to 20%, group II 14 patients superficially burned over 20%, group III 15 patients profoundly burned up to 20% and group IV 14 patients profoundly burned over 20%. First and second group of patients were treated conservatively with silver-sulfadiazine, and third and fourth with surgical method of early tangential excision. From all patients were collected blood samples (sera ) on 1st, 3rd, 5th, 7th, 14th, 21st and 28th day after injury for biochemical and hematological parameters, as well as oxidative stress parameters (TCA - total capacity of antioxidants and oLAb - autoantibodies against oLDL).

In vitro assays

Total Capacity of Antioxidants (TCA)

In the sera of patients we have determined total capacity of antioxidants by spetrophotometric method (TCA assay, Tatzber KEG, Austria). Kit was designed for determination of total unspecific antioxidative capacity of sera.

Autoantibodies against oxidized low density lipoproteins (oLAb)

In the samples of EDTA plasma we monitored changes in dynamics of the titer of autoantibodies against oxidized low density lipoproteins. This kit is ELISA made by Tatzber KEG, Austria (Schumacher et al. 1995, Borovic et al., 1995) .


54

Results

Total Capacity of Antioxidants

Results are shown in figure 1.

Fig 1. Shows average values of TCA (Total Capacity of Antioxidants) as equivalents to mg/ml uric acid standard for all four groups and control group during a period of 28 days.

There was no significant change of values in I group. Second group had significant changes on 1st and 3 rd day with average values of 0.0443mg/ml and 0.0449 mg/ml eq.u.a. (equivalent of uric acid ). Third group had significant changes on 3rd and on 5th day with average values 0.0481 mg/ml and 0.470 mg/ml eq.u.a. Group IV had significant changes on 1st , 3rd , 5th , 7th and 14th day with average values 0.0368 mg/ml; 0.0286 mg/ml; 0.0377 mg/ml; 0.0392 mg/ml and 0.0327 mg/ml consecutively. Average control value was 0.062mg/ml eq.u.a.

In all patients burn trauma had caused decrease of TCA, with lowest values in group IV. In first day diminution was 40,6 % comparing to control, and in third day 53,8% , which was the lowest value in general. At the end of the third week there were no more differences relating to control group. Considerable differences among groups were present on 14th day between group IV and other three groups .

Autoantibodies against oxidized low-density lipoprotein

For groups I, II and III is that there was no differences in the titer until 7th day (Fig. 2). Comparing to this three groups, group IV had significantly higher titer of the antibodies already on 5th day comparing to control, as well as comparing to values of the 1st day (p<0.005). There were no significant differences in values of the 1st and 3rd day between groups. Significant difference (p<0.005) was seen on 5th day between group IV and other three groups. All groups have also maximal titer on 14th day. The 4th group has on 5th day significant increase in antibody titer on 829 U (p<0.005) compared to control group (219U) and to previous values. On the 7th day mean value of the group IV was 1427U, and on 14th day there was a maximum of titer (1510U) in total and in particular group. After the 14th day the mean value was decreasing, on the 21st day the value was 1316U, and on 28th day it was 1226U.

There was no significant difference between first three groups on particular days. However, there was a significant difference (p<0.005) between group IV and other groups in total, in particular (p<0.0001) if days 5 to the day 28 were compared.

Fig.1. Value of Total Antioxidant capacity

Pk

PkPk

Pk

Pk,Pg I II III

PkPk

Pk

Pk

0

0,01

0,02

0,03

0,04

0,05

0,06

0,07

0,08

1 3 5 7 14 21 28

mg/

ml

I group

II group

III groupIV group

control group


55

Fig 2. Summarizes changes of average values of antibody titer against oLDL in period of 28 days Groups I, II and III had significant changes from 7th day with average values of 458 U, 596U and 792 U consecutively. On the 14th day all three groups had maximum values of 588 U, 794 U and 712 U. Group IV had significant changes on 5th day with average value of 829 U and max. Value on the 14th day with average value of 1510 U. Average control value was 212U. Pk significant change of some point comparing with control value. Pp significant change of some point comparing with beginning value (1st day value of each group). Pg and/or I II III IV significant change between each group

Discussion

Results have shown that oxidative stress was present in all groups of patients. There was an obvious correlation between depth and total burn surface and intensity of oxidative stress, or in other words, development of oxidative stress seems to be dependent on intensity of damage or quantity of burned tissue.

The highest intensity of peroxidation was observed in the group IV, but the increase was observed even in the group I (superficially burned) between 3rd and 5th day. Decrease in antioxidant capacity was biggest in profoundly burned patients, but there was also a significant decrease in TCA in groups II and III. Titer of antibodies against oLDL was highest in group IV (profoundly burned), but the increase was also present in group I, so this method could be clinically used as a good parameter to monitor oxidative stress in burns.

References Borovic S, Zarkovic, Wildburger R, Tatzber F, Jurin M. Post-traumatic differences in titer of autoantibodies against oxidized low density lipoprotein (oLDL) in the sera of patients with traumatic bone fractures and brain injury. Period biol 1995;97:289-93.

Schumacher M, Eber B, Tatzber F, Kaufmann P, Halwachs G, Fruhwald F M, Zweiker R, Esterbauer H, Klein W. Transient reduction of autoantibodies against oxidized LDL in patients with acute myocardial infraction. Free Radic Biol Med 1995;18:1087-91.

Stipancic I., Zarkovic N. Ucinci ozljede - operacije na funkciju imunoloskog sustava. Lijec. Vjes, 1997; 119:279-290

Virella G, Virella I, Leman RB, Pryor MB, Lopes-Virella MV. Anti-oxidized low-density lipoprotein antibodies in patients with coronary heart disease and normal healthy volunteers. Int J Clin Lab Res, 1993;23:95-101.

F ig . 2 .T itre o f antib ody ag ains t o LD L

Pk,PpPk,Pp

Pk,Pp

Pk,Pp,Pg IPk,Pp,Pg I

Pk

Pk,PpPk,Pp

Pk,Pp

Pk,Pp

Pk,Pp,Pg I II III

Pk,Pp,Pg I II III

Pk,Pp,Pg I II III

Pk,Pp,Pg I II III

Pk,Pp

0

200

400

600

800

1000

1200

1400

1600

1 3 5 7 14 21 28

I g roupII g roupII I g ro upIV g roupcontro l g roup

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Oxidative Stress and Lipid Peroxidation: Physiology and ...Mrs. Vjera Juric and collaborators from the Citizens' Society of Medjimurje Ms. Anja Kresic, Ms. Jelena Zenko. Ms. Silva