2003 39th meeting of the polish biochemical society 27 ... · 1 — katedra genetyki molekularnej,...

group and the epoxidation of the C=C double bond. Ex-amples will be presented for the interaction of HNE withvarious classes of biomolecules such as proteins and pep-

tides, lipids and nucleic acids and the biochemical conse-quences will be discussed.References:Poli G, Schaur RJ (2000) IUBMB Life, 50: 315–321.

44 Poster

Involvement of free radicals in the genotoxity of amoxicillin in human gastricmucosa cells and lymphocytes

Micha³ Arabski1, Maria Wiœniewska-Jarosiñska2, Pawe³ KaŸmierczak2, Józef Drzewoski3, Janusz B³asiak1

1 — Katedra Genetyki Molekularnej, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ, 2 — Pracownia Endoskopowa Szpitalaœw. Jana Bo¿ego, Uniwersytet Medyczny, £ódŸ, 3 — Zak³ad Farmakologii Klinicznej z Oddzia³em Klinicznym ChoróbWewnêtrznych, Uniwersytet Medyczny, £ódŸ

Amoxicillin is a penicillin derivative belonging to agroup of beta-lactam antibiotics used for treatment ofH. pylori infection. Clinical use of amoxicillin is under-lined by its antibacterial activity but an important ques-tion is whether and how amoxicillin interacts with DNAof human cells. We investigated the genotoxity ofamoxicillin in human peripheral blood lymphocytes(PBL), H. pylori infected (Hp+) and non-infected (Hp–)gastric mucosa cells (GMCs). GMCs were isolated frombiopsies obtained during gastroscopy. We used alkalinecomet assay to evaluate DNA damage and repair andwe employed formamidopyrimidine-DNA glycosylase(fpg) and endonuclease III homologue-1 (NTH) en-zymes recognizing oxidized DNA bases to assess oxida-tive DNA damage induced by amoxicillin. We appliedantioxidants and free radical scavengers: melatonin, vi-tamins C and E and nitrone spin trap PBN(N-tert-butyl-[�]-phenyl-nitrone) to assess free radicalsinvolvement in the genotoxity of amoxicillin.Amoxicillin (1–5 mM) induced dose-dependent DNAdamage in PBL, Hp+ and Hp– GMCs and the geno-

toxity of drug was more pronounced in Hp– than inHp+ GMCs. We showed that melatonin decreased theextent of DNA damage induced by amoxicillin in alltypes of cells. DNA damage induced by amoxicillin at 5mM was repaired after 60 min. However, cells exposedto 10 �M H2O2 were not able to complete repair DNAdamage during a 120 min incubation in the presence ofamoxicillin. Vitamins C and E reduced the extent ofDNA damage induced by amoxicillin in and PBL andHp+ GMCs. Cells exposed to amoxicillin and treatedwith enzymes recognizing oxidized bases (NTH, fpg)displayed greater extent of DNA damage that those nottreated with enzymes. The level of DNA lesions recog-nized by fpg glycosylase was higher in Hp+ GMC thanin PBL. The nitrone spin trap PBN reduced the extentof DNA damage in all types of cells. In conclusionamoxicillin induces DNA damage in PBL, Hp+ and Hp–GMC. The results obtained suggest that free radicalsmight be involved in the formation of DNA lesions in-duced by amoxicillin and antioxidant agents may be ap-plied to reduce DNA damage.

45 Poster

The effects of naturally occurring plant phenolics on the production of hydrogen per-oxide in mouse epidermis following treatment with the tumor promoter 12-O-tetra-decanoyl phorbol-13-acetate

Wanda Baer-Dubowska, Ma³gorzata Zieliñska, Hanna Szaefer

Katedra Biochemii Farmaceutycznej, Akademia Medyczna im. K. Marcinkowskiego, ul. Grunwaldzka 6, 60-780 Poznañ

Naturally occurring phenolic acids: protocatechuic,chlorogenic and tannic acids and trihydroxystilbene,resveratrol, were shown to inhibit multistagecarcinogenesis in animal models including mouse epi-dermis. Treatment of mouse skin with tumor promoter12-O-tetradecanoyl phorbol-13-acetate (TPA) induced

the production of hydrogen peroxide in keratinocytesas well as in skin-infiltrating leukocytes. In this studythe ability of plant phenolics to modulate the produc-tion of intracellular hydrogen peroxide was analyzed byflow cytometry. Following a single treatment of micewith TPA (6.8 nmol; 24 h) two subpopulations of epider-

2003 39th Meeting of the Polish Biochemical Society 27

Andrzej Basaj

group and the epoxidation of the C=C double bond. Examples will be presented for the interaction of HNE with various classes of biomolecules such as proteins and peptides, lipids and nucleic acids and the biochemical consequences will be discussed. References: Poli G, Schaur RJ (2000) IUBMB Life, 50: 315–321.

mal cells were identified, which oxidized 1.2-fold higherlevels of 2’,7’-dichlorofluorescin (DCFH) than cells iso-lated from mice treated with acetone alone. Pretreat-ment of mice with 16 �mol of phenolics 15 minutes be-fore TPA treatment decreased the production of hydro-gen peroxide in both subpopulations. The most effi-cient inhibitors in subpopulation 1 were tannic acidand resveratrol which reduced the production of hydro-gen peroxide by 16 and 15%, respectively (mean chan-nel intensity of oxidized DCFH green fluorescence141.0 ± 22.6 and 143.3 ± 14.2). In subpopulation 2, how-

ever, protocatechuic acid and chlorogenic acid weremore potent inhibitors decreasing the intracellular pro-duction of hydrogen peroxide by 21 and 35%, respec-tively (mean channel intensity of oxidized DCFH greenfluorescence 75.0 ± 15.5 and 61.7 ± 14.9). These resultssuggest that the anti-promotional effects of these struc-turally diverse phenolics might be partly explained byinhibition of the TPA-induced ROS formation, butmore detailed studies are necessary to characterizebetter this phenomenon.

46 Poster

Prooxidative effects of TEMPO in reactions with oxidants and glutathione

Aneta Balcerczyk, Miros³aw Soszyñski, Grzegorz Bartosz

Katedra Biofizyki Molekularnej, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ

Nitroxides, stable free radicals used as probes inESR, are gaining popularity as antioxidants. However,prooxidant effects of nitroxides have also been noted.We have found previously that 2,2,6,6-tetramethyl-piperidine-1-oxyl (Tempo) has a prooxidative effect aug-menting glutathione oxidation by peroxynitrite(G³êbska et al., Free Radic Bio Med, in press). Our pres-ent results demonstrate that this prooxidative effect ofTempo can also be observed in other in vitro systemswith a range of oxidants. While incubation of severaloxidants (ammonium persulfate, t-butyl hydro-peroxide, hydrogen peroxide and hypochlorite), 100�M, with 100 �M glutathione in Chelex-treated 100 mMphosphate buffer, pH 7.4 at 37°C for up to 3 hours,brought about some oxidation of glutathione, the oxida-tion of glutathione was much higher in the presence of100 �M Tempo which did not react with glutathione toa significant extent by itself. Oxidation of glutathionewas accompanied by decrease of the ESR signal ofTempo. This effect was attenuated in the presence of

100 �M EDTA, significant augmentation of oxidationof glutathione by Tempo, and loss of Tempo ESR signalbeing observed only for persulfate. Apparently, EDTApresent in equimolar amount with respect to other re-actants became involved in the reaction protectingTempo. We ascribe the prooxidative effect of Tempo byits participation in chain reactions with theglutathionyl radical formed by the oxidants. The occur-rence of such chain reaction involving free radical in-termediates may damage biological macromolecules.Indeed, inactivation of and loss of thiol groups in alco-hol dehydrogenase (100 �g/ml) exposed to 1 mM by2,2’-azobis(2-amidinopropane) (AAPH) at 37oC wasaugmented by 25–100 �M Tempo. Oxidation ofdeoxyribose (1 mM) to thiobarbituric acid-reactiveproducts by persulfate and t-butyl hydroperoxide (100�M) in the presence of glutathione (100 �M) was in-creased by Tempo (100 �M). These results point to apossibility of a prooxidant action of nitroxides in bio-logical systems in the presence of appropriate oxidants.

47 Poster

Is antimutagenic MTH1 protein a marker of oxidative stress?

Karol Bia³kowski1, Kazimierz Kasprzak2

1 — Katedra i Zak³ad Biochemii Klinicznej, Akademia Medyczna, ul. Kar³owicza 24, 85-092 Bydgoszcz, 2 — Laboratory ofComparative Carcinogenesis, National Cancer Institute at Frederick, National Institutes of Health, Bldg. 538, Rm. 205E, MD21702 Frederick, USA

Mammalian MTH1 protein is thought to beantimutagenic enzyme that prevents the incorpora-tion of oxidatively modified purine nucleotides into

nucleic acids. It decomposes a broad spectrum of ribo-nucleoside and 2’-deoxyribonucleoside 5’-triphospha-tes (NTPs and dNTPs) to the corresponding

28 Session 1. Biochemistry of oxidative stress 2003

nucleoside 5’-monophosphates and inorganic pyro-phosphate. Although canonical dNTPs and NTPs arealso substrates for MTH1 protein, the enzyme decom-poses most specifically the products of oxidative dam-age to purine dNTPs and NTPs (e.g. 8-oxo-dGTP,8-oxo-dATP, 2-OH-dATP, 2-OH-ATP, 8-oxo-GTP) thatmay cause point mutations or transcription errors af-ter the incorporation into DNA and RNA, respec-tively.

There is a growing evidence for overexpression ofMTH1 gene in human cancer cells. Higher levels ofMTH1 mRNA in human tumors and cancer cell lineshave been most frequently suggested to arise from anincreased exposure of cancer tissues to endogenous re-

active oxygen species. Moreover, some authors proposeto use that overexpression as a molecular marker of oxi-dative stress and/or carcinogenesis.

To test whether oxidative stress can indeed affectMTH1, we investigated the influence of ionizing radiation(source of oxidative stress) on the MTH1 8-oxo-dGTPaseactivity in mouse tissues. Male BL6 mice, 6-wk old, weresubjected to 0, 10, 25, 50, and 100 cGy of 137Csgamma-radiation, 100 cGy/min, given in a single dose perwhole body, and killed 4, 8, and 24 hrs later (three miceper each dose/kill time combination). The 8-oxo-dGTPaseactivity measurements were performed in livers. Our re-sults did not reveal any significant changes of the8-oxo-dGTPase activity following irradiation.

48 Poster

Does oxidative stress modify the activity of the erythrocyte sodium-protonexchanger?

Joanna Bober1, Ewa Kwiatkowska2, Karolina Kêdzierska2, Maria Olszewska1, Barbara Do³êgowska1, KazimierzCiechanowski2, Dariusz Chlubek1

1 — Katedra i Zak³ad Biochemii i Chemii, Pomorska Akademia Medyczna, ul. Powstañców Wlkp. 72, 70-111 Szczecin,2 — Klinika Nefrologii, Transplantologii i Chorób Wewnêtrznych, Pomorska Akademia Medyczna, ul. Powstañców Wlkp. 72,70-111 Szczecin

Reactive oxygen species (ROS) generated in patientswith chronic renal failure and during sessions ofhemodialysis exert an adverse action on the cellularmembrane of erythrocytes by interacting with mem-brane lipids and other structures, includingion-transporting systems like the sodium-protonexchanger (NHE). The activity of NHE results in the up-take of one sodium ion in exchange for one proton re-leased from the cell. ROS and other factors modifyNHE activity with the mediation of mitogen-activatedkinases. We have studied patients with chronic renalfailure (CRF) treated either (1) conservatively or withhemodialysis (HD) using (2) glucose-free (HD-g(–)) or(3) glucose-supplemented dialysate (HD-g(+)). The re-sults were compared with controls. Altogether, 87 sub-jects participated. The levels of thiobarbituricacid-reactive substances (TBARS) were determined toassess oxidative stress. NHE kinetics were establishedby measuring Vmax, Km, and activity at intracellular pHof 6.4 (VpHi 6.4). The effect of superoxides on the activ-ity of NHE in vitro was studied as well.

We found elevated levels of TBARS in all patientgroups. Following hemodialysis, TBARS levels werereduced in HD-g(+) (1.36 ± 0.30 vs. 1.17 ± 0.31

�mol/l) and increased in HD-g(–) patients (1.56 ±0.27 vs. 2.01 ± 0.55 �mol/l). The activity of NHE dif-fered in conservatively treated patients as comparedwith the remaining groups. While Km values re-mained essentially unchanged, Vmax and VpHi 6.4were markedly elevated. NHE activity (VpHi 6.4) wasunchanged after hemodialysis in HD-g(+) (8.43 ± 2.37vs. 8.95 ± 2.03 mmol H+/l RBC/h) and reduced inHD-g(–) patients (9.10 ± 3.17 vs. 6.59 ± 2.62 mmolH+/l RBC/h).

Low concentrations of the hydroxyl radical in vitrowere accompanied by significantly enhanced NHE ac-tivities (increase of more than 50% over baseline val-ues). Regardless of the concentrations of t-BOOH, NHEactivity remained lower in the glucose-free incubationmedium. This model appears relevant to CRF patientsin the sense that NHE activity in patients with moder-ately severe disease is enhanced, while terminal dis-ease is associated with normal activity prior tohemodialysis. Glucose-free dialysate produces substan-tial oxidative stress and suppresses NHE activity. Glu-cose supplementation results in a relatively minor oxi-dative stress with unchanged Vmax and reduced Km, re-flecting a form of activation of NHE.


49 Poster

Prehemolytic and hemolytic changes in erythrocytes incubated with catechol

Bo¿ena Bukowska, Sylwia Kowalska, Wirgiliusz Duda

Katedra Biofizyki Ska¿eñ Œrodowiska, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ

The effects of exposure of human erythrocytes to dif-ferent concentrations of catechol were studied. The in-vestigations concerned mainly the content ofglutathione (GSH, Ellman, 1959, and GSH + GSSG,Akerboom and Sies, 1981), activities of glutathionereductase (Carlberg and Mannervik, 1972), glutathioneS-transferase (Rice-Evans et al., 1991) and glu-cose-6-phosphate dehydrogenase (G-6-PDH; Sigma 345UV), hemoglobin oxidation and hemolysis.

Erythrocytes at a 5% hematocrit were mixed continu-ously at 37°C with 10 to 100 ppm catechol during 1 h.Control reagent samples of catechol with all other re-agents were also prepared and obtained correctionswere taken into account when appropriate. Student’spaired t-test was used; differences were considered tobe significant for p<0.05.

Catechol decreased the level of GSH (control, 1.22 ±0.10 mmol/l cells; 10 ppm catechol, 0.89 ± 0.35 mmol/l

cells) and total glutathione in erythrocytes (control,1.37 ± 0.18 mmol/l cells; 10 ppm catechol, 1.23 ± 0.24mmol/l cells; 50 ppm, 1.08 ± 0.22 mmol/l cells), signifi-cantly increased the activity of glutathione reductase(control, 2.28 ± 0.58 U/g Hb; 50 ppm catechol, 2.72 ±0.61 U/g Hb), glutathione S-transferase (control 5.00 ±0.96 U/g Hb; 100 ppm catechol, 5.66 U/g Hb ± 1.09 U/gHb) and glucose-6-phosphate dehydrogenase (control,6.29 ± 1.39 U/g Hb; 50 ppm catechol, 7.03 ± 0.90 U/gHb).

Incubation of hemoglobin with catechol increased thelevel of met-Hb (control, 1.85 ± 0.33% met Hb; 50 ppmcatechol, 7.21 ± 0.91% met-Hb). Catechol inducedhemolysis (control, 0.81 ± 0.18%; 10 ppm catechol, 1.27± 0.2%).

These effects of low doses of catechol may suggest afree radical mechanism of action of this compound onthe red blood cells.

50 Poster

Markers of oxidative damage in the blood of hypercholesterolemic children

Magdalena Che³chowska, Teresa Laskowska-Klita

Zak³ad Biochemii i Diagnostyki Laboratoryjnej, Instytut Matki i Dziecka, ul. Kasprzaka 17A, 01-211 Warszawa

Cardiovascular disease affecting adults have been es-tablished to begin in childhood. Hypercholesterolemicchildren have higher total and LDL cholesterol levels aswell as apoB and lower HDL cholesterol and apoA-Ithan normocholesterolemic ones. Oxidative modifica-tions of subendothelial lipoproteins (oxidation of LDL)is involved in the pathogenesis of atherosclerosis. Theaim of this study was to examine whether markers ofoxidative damage to protein (protein carbonyls) andlipids (malondialdehyde) may be modified inhypercholesterolemic children. We have found thatmean concentration of plasma protein carbonyls was0.76 ± 0.21 nmol/mg protein (range of 0.39–1.29nmol/mg protein) in hypercholesterolemic patientsand was higher by 20% than in the control (0.63 ± 0.09nmol/mg protein; p<0.05 ).

Plasma concentration of malondialdehyde (MDA)was 2.91 ± 0.61 �mol/l (range of 1.95–4.55 �mol/l). In

18 from 32 children studied, MDA level was elevatedabove the highest values observed in the healthy group(2.80 �mol/l; p<0.0001).

Total radical-trapping antioxidant parameters(TRAP) amounted to 616 ± 92 �mol/l and 733 ± 87�mol/l in hypercholesterolemic children and control,respectively. The difference was statistically significant(p<0.001).

The obtained results indicate that in hyper-cholesterolemic patients oxidative stress caused oxida-tive modification of proteins and peroxidation of lipidswhich was accompanied by diminished antioxidant pro-tection.

These results indicate that certain hypercholesterol-emic patients are susceptible to oxidative damagetherefore it seems that adequate and balanced diet isessential for achieving and maintaining normal anti-oxidant barrier in these subjects.


51 Poster

The effect of quinapril and perindopril therapies on the level of malondialdehydeand superoxide dismutase and catalase activities in erythrocytes of geriatric patientswith essential arterial hypertension

Jolanta Czuczejko1, Karolina Szewczyk-Golec1, Kornelia Kêdziora-Kornatowska2, Jadwiga Motyl2, HannaPawluk1, Katarzyna Kot³owska1, Józef Kêdziora1

1 — Katedra i Zak³ad Biochemii, Akademia Medyczna im. Ludwika Rydygiera, ul. Kar³owicza 24, 85-092 Bydgoszcz, 2 — Katedrai Klinika Geriatrii, Akademia Medyczna im. Ludwika Rydygiera, ul. M. Sk³odowskiej-Curie 9, 85-094 Bydgoszcz

The activation of the rennin-angiotensin system is in-volved in the pathogenesis of hypertension. Theprincipial effector peptide of this system is angiotensinII, which may participate in vascular pathology throughstimulation of superoxide anion (O2

–) and hydrogenperoxide (H2O2) formation. It has been suggested thatoverproduction of these reactive oxygen species by theendothelium, smooth muscle cells and adventitiousfibroblasts is involved in nitric oxide (NO) inactivation.In this conditions NO can be scavenged by O2

– to highlyreactive oxygen species — ONOO–. Additionally O2

– at-tacks membranes containing polyunsaturated fatty ac-ids and this initiates lipid peroxidation. The metabo-lites of lipid peroxidation, such as malondialdehyde(MDA), are cytotoxic and are capable of inactivatingmany cellular proteins. In the cells O2

– is dismutated toH2O2 by superoxide dismutase (SOD) and H2O2 is con-verted to water by catalase (CAT). The activities ofthese antioxidant enzymes are reduced in subjects withessential hypertension and in the course of aging. Thefall in SOD and CAT activities, increased ROS produc-tion and NO inactivation contributes to vascular injuryin hypertension.

We aimed to determine the effect of two angiotensinconverting enzyme (ACE) inhibitors: quinapril andperindopril on the malonylodialdehyde concentrationand the superoxide dismutase (SOD) and catalase(CAT) activities.

We measured the content of thiobarbituric-acid reac-tive substances (mainly malondialdehyde; TBA-RS, themethod of Placer et al.), SOD (the method of Misra andFridovich) and CAT (the method of Beers and Sizer) ac-tivities in erythrocytes of geriatric patients (mean age 74years) with essential arterial hypertension. The bloodwas taken before perindopril and quinapril treatment(day “0”) and after 7 (day “7”) and 45 days (day “45”) ofthe therapy. The MDA content in erythrocytes of the pa-tients decreased significantly after 45 days perindopriland quinapril treatment, from 0.34 ± 0.13 (day 0) downto 0.26 ± 0.06 �g/gHb (day 45; p<0.05). There was no sig-nificant difference in the remaining parameters betweenthe patients at different stages of the therapy. The SODactivity in erythrocytes treatment exhibited an increas-ing trend on day 45. Our results indicate that quinapriland perindopril may be useful to modify the ROS-relatedprocesses in hypertension.

52 Poster

Antioxidant effectiveness of acyl chain derivatives of 4-OH-TEMPO

Izabela Æwiklak1, Dorota BudŸko1, Micha³ WoŸniak1, Jerzy Nowak2, Takashi Wakabayashi3

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Katedra i Zak³adBiofizyki, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 3 — Department of Cell Biology and MolecularPathology, Medical University of Gdañsk, ul. Dêbinki 1, 80-211 Gdañsk

In the present study, effectiveness of newly synthe-sized TEMPOL derivatives (4-octanoyl- and 4-palmi-toyl-TEMPO) as scavengers of methyl radicals originat-ing from cytochrome P–450scc conversion of ButOOHin human term placental submitochondrial particle wasstudied. Both aminoxyls have been found to accumu-late inside the membrane as evidenced by ESR spec-troscopy. 4-Octanoyl-TEMPO displayed protective ef-fect against ButOOH induced oxidative stress while the

elongation of carbon chain up to C16 giving palmitoylderivative resulted in dramatic attenuation of aminoxyleffectivity.

Time dependent spin decay method confirmed highreactivity toward reactive oxygen species only ofweakly immobilized aminoxyl derivative, namely4-octanoyl-TEMPO. The results revealed that4-palmitoyl-TEMPO being strongly immobilized in bio-logical membranes became less potent antioxidant.


53 Poster

The effects of L-DOPA and dopamine on glucose formation in rabbit kidney-cortextubules

Jakub Dro¿ak1, Katarzyna Chodnicka1, Jadwiga Bry³a2

1 — Zak³ad Regulacji Metabolizmu, Uniwersytet Warszawski, ul. Miecznikowa 1, 02-089 Warszawa, 2 — Zak³ad RegulacjiMetabolizmu, Wydzia³ Biologii, Uniwersytet Warszawski, ul. Miecznikowa 1, 02-096 Warszawa

L-3,4-Dihydroxyphenylalanine (L-DOPA) is so far themost effective drug in the treatment of Parkinson’s dis-ease. It has been reported that circulating L-DOPA isextracted by kidney and converted to dopamine, consid-ered as an autocrine-paracrine substance regulatingmany renal functions. Moreover, degradation of bothL-DOPA and dopamine is accompanied by generatinghydrogen peroxide (H2O2), which may disturb cellularmetabolism.

The aim of the present investigation was to study theeffect of L-DOPA (100–500 microM) and its breakdownproducts on glucose synthesis in kidney-cortex tubulesincubated with pyruvate, dihydroxyacetone, alani-ne+glycerol+octanoate or aspartate+glycerol+octa-noate. Both dopamine and L-3,4-dihydroxyphenylaceticacid (DOPAC) appeared to be the only products ofL-DOPA degradation. The formation of homovanillicacid (HVA) was not detected. A rapid intracellular deg-radation of 100 microM L-DOPA was accompanied by adecrease in glucose formation from pyruvate, dihydro-xyacetone, aspartate+glycerol+octanoate (74.98 ± 9.33to 59.24 ± 7.01, P<0.005; 45.99 ± 7.14 to 27.83 ± 7.52,P<0.005; 103.54 ± 7.96 to 78.5 ± 10.41, P<0.005micromol x h–1 x mg–1 dry weight, respectively) but not

from alanine+glycerol+octanoate (63.16 ± 6.87 to 58.53± 4.64 micromol x h–1 x mg–1 dry weight). Dopamine af-fected glucose production similarly. The degree ofgluconeogenesis inhibition was proportional toL-DOPA or dopamine concentration and in the pres-ence of 500 microM L-DOPA, glucose production fromalanine+glycerol+octanoate was significantly dimin-ished (63.16 ± 6.87 to 35.98 ± 4.27 micromol x h–1 xmg–1 dry weight, p<0.05). Inhibition of dopamine deg-radation by Selegiline (Deprenyl), an inhibitor ofmonoamine oxidase B (MAO B) currently available forthe treatment of Parkinson’s disease, attenuated inhib-itory effects of both L-DOPA and dopamine on the rateof gluconeogenesis, while DOPAC, accumulating in theextracellular medium during L-DOPA breakdown, didnot have any influence on glucose synthesis. In addi-tion, 100 microM L-DOPA significantly diminished theintracellular glutathione (GSH) content (1.63 ± 0.04 to1.20 ± 0.10 micromol x mg–1 dry weight p<0.05) in thetubules incubated with pyruvate. In view of these datait seems likely that L-DOPA metabolism might induceoxidative stress resulting in a decrease in glucose for-mation in rabbit renal tubules.This investigation was supported by a departmental grant.

54 Poster

Glucose modulates nitric oxide-dependent vascular endothelial growth factorsynthesis

Józef Dulak1, Katarzyna Tomala1, Urszula Jarosz1, Alicja Józkowicz2

1 — Department of Cell Biochemistry, Faculty of Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków,2 — Department of Molecular Genetics and Genetic Engineering, Jagiellonian University, ul. Granostajowa 7, 30-387 Kraków

Background: Synthesis of vascular endothelialgrowth factor (VEGF), the major angiogenic mediator,is enhanced by nitric oxide (NO) (Dulak et al,Arterioscler Thromb Vasc Biol. 2000, 20: 659–666;Józkowicz et al., Cardiovasc Res, 2001, 51: 773–783)and depends on the activity of heme oxygenase-1(HO-1), the enzyme degrading heme to iron, carbonmonoxide and biliverdin (Dulak et al., Antioxid RedoxSignal, 2002, 4: 229–240; Józkowicz et al., Antioxid Re-dox Signal, 2002, 4: 577–585). Interruptions in the re-ciprocal relationship between NO and VEGF synthesismay thus underlie the disturbances of angiogenesis ob-

served in atherosclerosis and diabetes. Addressing thisproblem we have tested the effect of glucose on NO andVEGF synthesis. Methods: Rat vascular smooth musclecells (VSMC) were stimulated with IL-1� and TNF-� inmedia containing 5.5 mM or 25 mM glucose. NO gener-ation was determined by nitrite measurement usingGriess method, VEGF production was measured byELISA. VEGF, HO-1 and inducible NO synthase (NOSII) expression was determined by RT-PCR and VEGFpromoter activity was checked by a reporter gene assayin cells transfected with VEGF-SEAP reporter plasmid.Results: Cytokines induced VEGF synthesis, the level


of which was dependent on the amount of generatedNO. This effect occurred in cells cultured in 5.5 mM glu-cose, while 25 mM glucose impaired induction of NOSII expression and NO generation, what resulted in at-tenuation of VEGF promoter activation and inhibitionof VEGF protein synthesis. Interestingly, HO-1 expres-

sion was also lower in the presence of 25 mM glucose.Conclusions. NO-dependent VEGF synthesis can be sig-nificantly attenuated by high glucose. The effect of glu-cose may thus contribute to the disturbances ofangiogenesis in diabetes and atherosclerosis.Supported by grants 3 PO4A 049 22 and 6 P04B 013 from the

State Committee for Scientific Research (KBN).

55 Poster

Antioxidant rescue of Saccharomyces cerevisiae �sod mutant sensitivity to superoxideradicals

Dorota Dziadkowiec1, Anna Krasowska1, Marcin £ukaszewicz1, Karolina Wojtowicz1, Karel Sigler2

1 — Instytut Genetyki i Mikrobiologii, Uniwersytet Wroc³awski, ul. Przybyszewskiego 63-77, 50148 Wroc³aw, 2 — Institute ofMicrobiology, Czech Academy of Science, Prague, Czech Republic

The growth of S. cerevisiae �sod1 strain lacking cyto-plasmic Cu,Zn-superoxide dismutase, �sod2 strain de-leted for mitochondrial Mn-superoxide dismutase and adouble mutant (�sod1�sod2) on glucose and respira-tory substrates (glycerol, lactate) was studied in thepresence and absence of atmospheric oxygen, in thepresence of the superoxide producer paraquat and/ornatural antioxidants (vitamin A, vitamin E, quercetin,�-carotene). The �sod1 and �sod1�sod2 mutants dis-played strongly reduced aerobic growth on glucose,glycerol and lactate. Surprisingly, �sod1�sod2 mutantgrew better aerobically on glucose; the additional dele-tion in mitochondrial SOD thus appears to trigger anunknown process decreasing cell sensitivity to oxygen.On mineral GO medium both strains failed to grow aer-obically in the absence of Met and Lys and the growthwas largely rescued by addition of these amino acids.

Paraquat inhibited their growth in a concentra-tion-dependent manner under anaerobiosis, the doublemutant exhibiting again lower superoxide sensitivity.Vitamins A and E at 1 �M rescued the oxygen-inducedgrowth defect of �sod1 strain but had no effect at 25�M; by contrast, in the �sod1�sod2 both vitaminswere effective even at the higher concentration.Quercetin also alleviated the growth defect of both mu-tants; its action depended on the aeration intensity andwas stronger against O2 than against paraquat. �-Caro-tene had no growth-restoring effect.The work was supported by Polish grants 3T09B 081 and

4T09B07922, the Grant Agency of the CR Academy ofSciences (grant S5020202), Czech Ministry of Education(grants CZE 01-032 and ME577), the Institutional Re-search Project AV0Z5020903 and grant 23/40 of theCzech-Polish Treaty on Scientif ic and Scien-tific-Technical Cooperation.

56 Poster

Catechol estrogen displays protective effect on albumin — important extracellular an-tioxidant being exposed to peroxyl radical generating system

Adam Figarski1, Piotr Walczak1, Jerzy Szymendera1, Teresa Powierza1, Lucedio Greci2, Micha³ WoŸniak1

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Dipartamento diScienza dei Materiali e della Terra, Universita di Ancona, Ancona, Italy

Albumin, a relatively small (MW 69,000) highly watersoluble protein and blood transporter of a wide range ofwater insoluble substances (e.g. fatty acids andestrogens) has been recently found to prevent neuronsagainst detrimental effect of stroke (Belayev, Stroke,2001). Protective effect of albumin can be ascribed toits antioxidative function.

Albumin has specific binding site for highly red-ox ac-tive copper ions and high percent of non-celuroplasmin

copper can exist firmly bound to albumin. Brain tissuedisplays efficient conversion of estrogen into catecholestrogens, very efficient scavengers of reactive oxygenspecies. Our results show that albumin at concentra-tion less than physiological can inhibit markedly cop-per-stimulated peroxidation of liposomal membranes.Albumin itself is attacked by radicals and largely dam-aged. Exposition of albumin solution (2.5 mg/ml) toperoxyl radical generating system (20 mM ButOOH, 5


mg PbO2) resulted in accumulation as high as 20 nmolof protein carbonyls/mg protein after 30 min of incuba-tion. 2-OH-E2 (10 �M) effectively prevented carbonylgroup formation while estradiol E2 at concentration

10-fold higher failed to display preventive effect.Quinolinic aminoxyl electron paramagnetic probe con-firmed different reactivity of E2 and 2-OH-E2 withperoxyl radicals.

57 Poster

Mechanism of peroxynitrite interaction with cytochrome c

Lidia Gêbicka, Joanna Didik

Miêdzyresortowy Instytut Techniki Radiacyjnej, Politechnika £ódzka, ul. Wróblewskiego 15, 93-590 £ódŸ

Kinetics of the reaction of peroxynitrite with ferriccytochrome c in the absence and presence of bicarbon-ate was studied. It was found that heme iron in ferriccytochrome c does not react directly with peroxynitrite.The rates of absorption changes in the Söret region ofcytochrome c caused by peroxynitrite or peroxy-nitrite/bicarbonate were the same as the rate of sponta-neous isomerization of peroxynitrite or, as the rate ofthe reaction of peroxynitrite with bicarbonate, respec-tively. It means that intermediate products ofperoxynitrite decomposition, �OH/�NO2 or, in the pres-ence of bicarbonate, CO3

–�/�NO2, are species responsi-ble for absorption changes in the Söret band ofcytochrome c. Modifications of heme center ofcytochrome c by radiolytically produced radicals, �OH,

�NO2 or CO3–� were also studied. Absorption changes

in the Söret band caused by �OH or CO3–� produced

radiolytically were much more significant that thoseobserved after peroxynitrite treatment, compared un-der similar concentrations of radicals. �NO2 producedradiolytically did not interact with heme center ofcytochrome c. Cytochrome c exhibited increasedperoxidase-like activity after reaction withperoxynitrite, as well as with radiolytically produced�OH, �NO2 or CO3

–� radicals. It means that modifica-tion of protein structure: oxidation of amino acidsand/or tyrosine nitration, facilitates reaction withH2O2 with heme iron of cytochrome c, followed by thereaction with substrate.

58 Poster

Caspase-3 activation in human osteosarcoma 143B cells proceeds through an earlycell membrane fluidity perturbation

Aleksandra Gil1, Anna Trzmiel1, Edyta Niemczyk2, Jakub Kêdzior1, Jan Spodnik3, Narcyz Knap1, TakashiWakabayashi2, S³awomir Œwirydowicz4, Micha³ WoŸniak1

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Department of CellBiology and Molecular Pathology, Medical University of Gdañsk, ul. Debinki 1, 80-211 Gdañsk, 3 — Katedra Anatomii iNeurobiologii, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 4 — Katedra i Zak³ad Biofizyki, AkademiaMedyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk

2-Methoxyestradiol (2ME) is an estrogen metaboliteformed by hydroxylation of estradiol followed by subse-quent O-methylation in the liver. Our flow cytometryanalysis revealed that 10 �M 2ME inhibited cell cyclecausing G2/M arrest. EPR measurements with5-doxylstearate revealed that 2ME perturbed stabilityof cell membrane as early as after 2 hrs of incubation byincreasing membrane microviscosity. On the otherhand after 4 hrs of incubation of osteosarcoma cell with2ME stiffness of cell membrane was switched into

destabilization, namely fluidization of lipid part of themembrane. Those effects were followed by induction ofsuperoxide production measured by dihydroethidiumstaining after 4 hrs of cells incubation with 2ME. Fol-lowing oxidative stress after 12 hrs of incubation, sig-nificant elevation of caspase-3 activity was observed.Altogether our result convey the idea that upon interac-tion with plasma membrane, membrane destabilizingproperties of 2ME provide an early molecular event ofproapoptotic signaling of 2ME.


59 Poster

Damage to erythrocyte membrane proteins by X-rays under anaerobic conditionsand in the presence of oxygen

Marta Gonciarz-Wach, Zofia Szweda-Lewandowska, Krzysztof Kwapisz

Katedra Biofizyki Molekularnej, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ

The aim of the present paper was to study the effectsof primary products of water radiolysis, mainly �OHradicals, on erythrocyte membrane proteins. In orderto study these effects the erythrocyte suspensions in0.1 M Na-phosphate buffer (hematocrit 1%) were irradi-ated from an X-ray source under air, N2O or argon,with prehemolytic irradiation doses.

Protein conformational changes were measured us-ing a maleimide spin label (MSL) and the content of–SH groups was determined in the range prehemolyticdoses (from 0.1 kGy to 1.6 kGy). The EPR spectra oferythrocyte ghosts labeled with MSL have shown the si-multaneous presence of strongly (S) and weakly (W)immobilized components. The W/S ratio and the con-tent of thiol groups decreased as a function of dose forerythrocytes irradiated under air as well as under N2Oor argon. At a dose of 1.6 kGy the W/S ratio fell downto 46.2 ± 3.4%, 52.9 ± 3.2% and 61.8 ± 3.2% of the con-trol value for erythrocytes irradiated under air, N2O

and argon, respectively. Similar changes have been ob-served in the content of thiol groups of membrane pro-teins. The amount of –SH groups decreased most sig-nificantly in cells irradiated under air, from 80.0 ± 2.1down to 38.7 ± 3.8 nmol/mg protein. The decrease inradiosensitivity under N2O and argon was correlatedwith the presence of protein aggregates in the mem-brane (15.8 ± 2.5% and 15.1 ± 2.7%, respectively), as es-timated by SDS/PAGE electrophoresis. The aggregateswere formed principally by –S–S– bridges. It can besuggested the cross-linking induced mainly by �OH rad-icals under anaerobic conditions is the main cause ofdecrease in erythrocyte sensitivity to hemolysis.

Postradiation hemolysis took place about two timesslower in erythrocytes irradiated under N2O than un-der air. However, erythrocytes irradiated under argonhemolysed ten times more slowly than those irradiatedunder air and four times more slowly than those irradi-ated under N2O.

60 Poster

Luminescence and electrochemical measurements of the free radical generation inplant defence response to fungal infection

Zbigniew Górski1, Jolanta Floryszak-Wieczorek2, Grzegorz Milczarek1, Aleksander Ciszewski1

1 — Institute of Chemistry and Technical Electrochemistry, Poznañ University of Technology, ul. Piotrowo 3, 60-965 Poznañ,2 — Department of Plant Physiology, Agricultural University, ul. Wo³yñska 35, 60-637 Poznañ

Luminescence leaf images of ivy-leaved pelargonium(Pelargonium peltatum hort.) pointwisely inoculatedwith grey mould (Botrytis cinerea) spores were analysedin this paper.

Measuring techniques of low invasiveness were used inthis study. Photometric results were obtained by a 16-bit“Night Owl” CCD camera (EG&G Berthold). The imageanalysis was carried out using WinLight (EC&G Berthold)and FCT (Poznañ University of Technology) programs.

Electrochemical measurements of NO were done usingthe Autolab potentiostat (ECO Chemie, Netherlands) ina three-electrode arrangement, with a Ag/AgCl as thereference and platinum wire as the counter electrode.The working electrode was platinum wire (20 �m) modi-fied with polymerized vanillin porphyrin.

The fluorescence intensity calculated from the wholeinfected leaf area showed a stable increased level in re-

lation to the emission level of a healthy leaf not sub-jected to the infection. The emission observed on thesuccessive days of grey mould development differedsignificantly from the ultraweak photon emission, as tothe intensity and range, in the spots and furthersurrounding of disease spots.

Moreover, we have also tried to find a correlation be-tween the observed changes in the luminescence level andnitric oxide generation during grey mould infection. Ni-tric oxide concentration was measured with the use ofmicroelectrodes, counted on the basis of the height ofNOx signal peaks, which were the highest in the furthersurrounding of diseased spots and in the infected leafnerves. The amount of NO generated by tissuesurrounding disease lesions was 0.80 �M and that pro-duced by the nerves of affected leaf was 0.58 �M. Healthyleaves did not show even trace amounts of nitric oxide.


61 Poster

Changes in prooxidative state within V79 cells induced by thiram in vitro

Emilia Grosicka, Bo¿enna Sadurska, Maria Szumi³o, Iwonna Rahden-Staroñ

Katedra i Zak³ad Biochemii, Akademia Medyczna, ul. Banacha 1, 02-097 Warszawa

Thiram is a reactive compound with strongmetal-binding characteristics, capable to interact with pro-tein and nonprotein thiol groups. Its toxicity is thought tobe mediated via induction of lipid peroxidation. However,oxidative damage of cellular proteins, although not so wellstudied, may also contribute to its toxicity, as well as to awide range of conditions in which roles of oxidative injuryhave been invoked, e.g. aging, oxygen toxicity and neuro-degenerative diseases. The most commonly used markerof protein oxidation are protein carbonyl groups, becauseprotein oxidation often (but not always) results in the for-mation of new carbonyls of arginine, lysine, threonine, orproline residues, and also because protein carbonyls canbe easily detected by hydrazone derivatization of carbonylgroups with an appropriate hydrazine reagent.

In the present work the toxic effect of thiram was investi-gated in cultured Chinese hamster fibroblasts (V79 cells).

Cells in exponential growth (600–1000 cells per 94 mmdishes) were exposed to thiram (0.1–50 �M) for 1 hour.Cell survival assays demonstrated that thiram induced adecrease in the recovery of viable cells: 80 ± 4% of the cellssurvived at a 1 �M concentration of fungicide. No cells sur-vived at a 100 �M concentration. Thiram exposure re-sulted in a rapid depletion of intracellular reducedglutathione. Thiram affected also the carbonyl group con-tent. The increase of carbonyl group content observed dur-ing 1 h treatment was dose-dependent (80 ± 2% at 50 �Mthiram).

The results indicate that thiram-induced cytotoxicity invitro is accompanied by a rapid depletion of intracellularreduced glutathione content with consecutive protein oxi-dation.

62 Poster

Assay of concentration rate of physiological vitamin C forms in blood plasma of pa-tients with larynx cancer

Krzysztof Grzegorczyk1, Maciej Rutkowski2, Alicja Lipka-Kociszewska3, Maria Kopff4

1 — Clinics of Gastroenterology of 5-th Clinical Hospital, Medical University in £odz, Pl. Hallera 1, £ódŸ, 2 — Chemistry andClinical Biochemistry Department, Medical University in Lodz, £ódŸ, 3 — Department of Laryngology of the Pirogows DistrictSpecialized Hospital in £odz, Medical University in £oŸ, ul. Wólczañska 191, £ód¿, 4 — Zak³ad Chemii i Biochemii KlinicznejWydzia³ Fizjoterapii, Uniwersytet Medyczny w £odzi, Pl. Hallera 1, 90-647 £ódŸ

Vitamin C is a hydrophilic antioxidant for the body,physiologically present in the reduced form (ascorbic acid,AA), able to deactivate reactive forms of oxide and in theoxidized form created in the process (dehydroascorbicacid, DA). Concentration ratio of those compounds is oneof the biomarkers of oxidative stress that may cause celldamages — which is an important factor in ethiopathologyof numerous diseases, including cancer.

The aim of this study was to assay concentration ratio ofboth vitamin C forms in blood plasma taken from patientssuffering from larynx cancer recognised both clinicallyand histopathologically.

The study was conducted on 133 subjects (males and fe-males) divided into four groups: I (control) — 33 healthysubjects, II — 33 patients with chronic tonsillitis, III — 33patients with precancerous state of larynx, IV — 34 pa-tients with larynx cancer. None of the subjects smoked ortook vitamins.

Blood was collected from cephalic vein on potassium oxa-late as a anticoagulation agent from subjects in fasting state.

Immediately after blood cells centrifugation concentrationsof both forms of vitamin C were assayed in the blood plasma.Assay was carried out with our own phosphotungstatemethod. Determinations were carried out with use ofLambda 14P (Perkin-Elmer) spectrophotometer. Basing onthe results of the analysis concentration ratios DA:AA werecalculated for all participants. Statistical analysis was per-formed with Student’s t-test.

Basing on the investigation the highest DA:AA concen-tration ratio was found for patients from the IV group.This may indicate increased prooxidative activity duringlarynx cancer, and the process probably leads to increasedconsumption of ascorbic acid.

Results of the study show significant diagnostic value ofthe assay of DA:AA concentration ratio in blood plasmafor patients with precancerous states and cancers, provid-ing clinically significant data concerning progression ofthe disease.The study has been carried out within WAM BW/40/2002

grant, and UM 502-17-019.


63 Poster

Antioxidant status in human brain intoxicated by heroin and psychotropic drugs

Marzena Gutowicz, Maria Pokorska-Lis, Sebastian Szymczakowski, Anna Barañczyk-KuŸma


Lipid peroxidation is considered as an important fea-ture of cellular injury. Since brain contains a largeamount of polyunsaturated fatty acids, it is especiallyvulnerable to oxidative stress, which has been impli-cated in many disorders including neurodegenerativeand cancerous diseases. The rate and extent ofperoxidation is reduced by endo- and exogenous antiox-idants.

In present study we determined activity and level ofendogenous enzymatic and nonenzymatic antioxidantssuch as catalase (CAT), glutathione peroxidase(GSH-Px), glutathione-S-transferase (GST), and re-duced glutathione (GSH).

Studies were conducted on different regions of hu-man brain (frontal, temporal, parietal and occipital cor-

tex, hippocampus, brain stem, white matter). Brain tis-sues were obtained from autopsy of young people whodied of heroin or intoxication with psychotropic drugsand who died suddenly, without any intoxication (thecontrol group).

The results showed that heroin intoxication leads todecrease in both GST activity and GSH level, whereaspsychotropic drugs intoxication increases GSH-Px ac-tivity and significantly decreases CAT activity. GSH-Pxand CAT activities are unchanged in heroin intoxicatedbrain, and GST activity is normal in intoxication withpsychotropic drugs. Thus, heroin and psychotropicdrugs lead to various changes in enzymatic barrierwhich protects brain against toxic compounds.

64

Production of DNA strand breaks by PMA in human neutrophils and inhibition bygallic acid

Ewa Ignatowicz1, Patrycja Piechocka1, Karolina Górecka2

1 — Katedra Biochemii Farmaceutycznej, Akademia Medyczna im. K. Marcinkowskiego, ul. Grunwaldzka 6, 60-780 Poznañ,2 — Instytut Genetyki Cz³owieka, Polska Akademia Nauk, ul. Strzeszyñska, 60-479 Poznañ

Oxidative stress leading to increased levels of reac-tive oxygen species (ROS) may induce oxidative DNAand protein damage. In aerobic organisms ROS produc-tion is strictly controlled, however inflammation andaccompanying reactions stimulate abnormally highROS synthesis and release from polymorphonuclearneutrophils and macrophages. Dietary polyphenolspossess antimutagenic and/or anticarcinogenic activi-ties which might be related to prevention of radical pro-duction and scavenging. In this study the effects of gal-lic acid on hydrogen peroxide production and DNA andprotein oxidation were assessed in humanpolymorphonuclear neutrophils. Cells were isolatedfrom venous blood and preincubated with gallic acid

(10 microM and 100 microM), followed by the stimula-tion to “oxidative burst” by phorbol myristate acetate.The generated hydrogen peroxide was measured in thephenol red reduction assay and H2O2-induced DNAstrand breaks were observed as alkaline single cellcomet assay. Protein oxidation products were mea-sured as carbonyl groups concentration, afterderivatization with dinitrophenylhydrazine. Gallic acidgave a marked inhibition of hydrogen peroxide produc-tion in PMN and reduced the formation of DNA strandbreakage in these cells, evoked by phorbol ester. Thelevel of carbonyl groups was also diminished. These re-sults indicate that simple phenolic acid may prevent theoxidative damage of cellular macromolecules.


65 Poster

Effect of postoperative treatment on serum zinc and alpha-2-macroglobulin concen-trations in men with chronic arterial occlusion

Maria Iskra1, Wac³aw Majewski2, Anna Pioruñska-Miko³ajczak3, Krzysztof Wiktorowicz4, Danuta Bara³kiewicz5

1 — Zak³ad Chemii Ogólnej, Katedra Biochemii Klinicznej, Akademia Medyczna, ul. Grunwaldzka 6, 60-780 Poznañ, 2 — KlinikaChirurgii Ogólnej i Naczyñ, Akademia Medyczna, ul. D³uga 1/2, 61-848 Poznañ, 3 — Department of General Chemistry, Chair ofChemistry and Clinical Biochemistry, University of Medical Sciences, ul. Grunwaldzka 6, 60-780 Poznañ, 4 — Department ofBiology and Environmental Sciences, University of Medical Sciences, ul. D³uga 1/2, 61-848 Poznañ, 5 — Department of Waterand Soil Analysis, Faculty of Chemistry, Adam Mickiewicz University, ul. Drzyma³y 24, Poznañ

Zinc can influence processes associated with oxida-tive stress, but has rather indirect antioxidant func-tions. Some its possible actions include restriction ofendogenous free radical production, contribution to thestructure of Cu,Zn-superoxide dismutase, maintenanceof tissue concentrations of metallotionein and stabiliza-tion of membrane structure. Many enzymes and pro-teins other than enzymes require Zn(II) ions at the ac-tive side. Some nonspecific proteins are responsible forthe transport of Zn in plasma: albumin (60–65%),transferrin (12%), and alpha-2-macroglobulin (MG)(20–30%). MG constitues a nonexchangeable fractionof Zn, is an inhibitor of matrix metalloproteases and be-longs to the acute phase proteins (APhPs). Prolongedstate of ischemia due to atherosclerosis, inflammationor surgery, may cause modifications in the levels of Znand Zn-related proteins. The study was aimed at anevaluation of the relationship between total Zn and MGconcentrations in serum of men with chronic arterialocclusion of lower limbs before surgery and during thepostoperative treatment.

The studied group consisted of men with chronic arte-rial occlusion of lower limbs treated surgically (vascu-lar reconstruction). Zn and MG concentrations wereanalyzed in serum before surgery and in the postopera-tive treatment (after 1–4, 7–12 and more than 12days). Zn was determined by atomic absorption spec-trometry, and MG by formation of immune complexesand the spectrophotometry.

The mean concentration of Zn, but not MG, wasfound increased in 7–12 days and after 12 days of treat-ment as compared to the value obtained before surgery(13.7 ± 2.1, 13.5 ± 2.9, 11.1 ± 2.2, respectively). Changesof Zn and MG in the postoperative treatment, calcu-lated as Cx — C0 , were negatively correlated with C0(r=0.7, p<0.05).

The results suggests that the exchangeable fraction ofZn (not bound to MG) in serum may participate in thedefence against deleterious effects of the oxidativestress due to atherosclerosis and surgery.

66 Poster

New method of detection of the carbon dioxide evolution from pyruvate scavenginghydrogen peroxide

Dagmara Jacewicz1, Aleksandra D¹browska1, Bogdan Banecki2, Micha³ WoŸniak3, Lech Chmurzyñski1

1 — Department of Chemistry, University of Gdañsk, ul. Sobieskiego 18/19, Gdañsk, 2 — Department of Molecular and CellularBiology, University of Gdañsk, Gdañsk, 3 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1,80-211 Gdañsk

Recent studies have shown that under conditions ofmyocardial ischemia pyruvate acts as a scavenger offree radicals produced by oxidative stress affording cer-tain quantities of H2O2 [1]. Most probably, pyruvic acidundergoes alternative transformation to CO2 and ace-tate upon reaction with H2O2, which is of cyto-protective importance [2–4]. Unfortunately, the mech-anism of the anti-oxidative action of the pyruvate hasnot been ultimately clarified to date.

As a model system we investigated the carbon dioxideuptake kinetics of [Cr(C2O4)(MADAH)(OH2)2]

+ ion con-taining bidentate sugar ligand, methyl 3-amino-2,3-di-

deoxy-�-arabino-hexopyranoside (MADAH) using thestopped-flow spectrophotometry method. It seemedthat a system consisting of a metal ion and a bioactivecompound could represent a reaction model to study anuptake CO2 captured in vivo from pyruvate in identicalway as that occurring in biological systems.

The results of investigations enabled to conclude thatthe studied reaction was proceeded in two steps [5–8].In the first rapid step, CO2 was captured to form an in-termediate complex in which the carbonate ion waslinked to Cr(III) through one oxygen atom. In the sec-ond, slower step, the final product was formed from the


intermediate product, in which the carbonate ion wasattached to the chromium cation through two oxygenatoms.

The results of research performed in this study may beuseful for elucidation of an enzymatic reaction pro-ceeded in organic matter, for instance the uptake and re-lease of CO2 catalyzed by pyruvate dehydrogenase(PGH) [9]. This enzyme catalyzes oxidative decarbo-xylation of pyruvate to acetate “activated” during thecourse of the enzymatic reaction by binding to CoA to af-ford acetyl-CoA. The activity of the pyruvatedehydrogenase complex is controlled by reversiblephosphorylation [10]. Under condition of energy short-age (low level of ATP), the pyruvate can undergo furthercatabolic transformations to acetyl-CoA, reduction tolactate or, at higher level of ATP can provide substratefor the synthesis of sugars, amino acids and fatty acids.

Finally, elucidation of the mechanism and elabora-tion of a method of CO2 uptake would pave the way to

evaluation of the pyruvate reaction with H2O2 and thepossibility of measurement of CO2 released during thisreaction.References:1. Crestanello JA, Lingle DM, Millili J, Whitman GJ (1998)

Surgery, 124: 92.2. Cavallini L, Valente M, Rigobello MP (1990) J Mol Cell

Cardiol, 22: 143.3. Bunger R, Swindall B, Brodie D (1986) J Mol Cell Cardiol,

18: 423.4. Mentzer RM, Van Wylen DG L, Shodi J (1989) Ann Surg,

209: 629.5. Dasgupta TP, Harris GM (1975) J Am Chem Soc, 97: 1733.6. Dasgupta TP, Harris GM (1977) J Am Chem Soc, 99: 2490.7. Dasgupta TP, Harris GM (1971) J Am Chem Soc, 93: 91.8. Dasgupta TP, Harris GM (1974) Inorg Chem, 13: 1275.9. Devli TM (1992) Textbook of Biochemistry with Clinical

Correlations, Wiley-Liss Inc., New York, 249.10. Huang B, Gudi R, Wu P, Harris RA, Hamilton J, Poper

KM (1998) J Biol Chem, 273: 17680.

67 Poster

Changes of electrical and luminescent parameters in Characae cells when local anes-thetics affect their metabolic state

Anna Jaœkowska1, Zbigniew Górski2, Andrzej Dudziak1, Robert Borc1, Ma³gorzata Gospodarek1, EdwardŒpiewla1

1 — Instytut Fizyki, Politechnika Lubelska, 20-618 Lublin, 2 — Institute of Chemistry and Technical Electrochemistry, PoznañUniversity of Technology, ul. Piotrowo 3, 60-965 Poznañ

The study examined electrical and luminescence re-sponses of Characeae internodal cells to the action oflocal anesthetics: procaine and lidocaine. Theelectrophysiology of these plants is well-known. Localanesthetics are characterized by the reduction or totalelimination of a membrane excitability. This propertyhas also been confirmed for plant cell membranes. It isremarkable that, when exposed to procaine orlidocaine, the cells do not react with an increased inten-sity of ultraweak luminescence (UWL) for 1/2 hour.They do react, however, when exposed to other stress-ors, e.g. injury, osmotic stress, temperature increaseetc.

Within 1/2 hour after the action of procaine (10–15mM) the amplitude and the frequency of occurrence ofaction potentials decrease. After that time the mem-brane loses its ability to generate action potentials. It islikely that this loss corresponds to the absence of a typi-cal increase in UWL intensity. In a natural situation inthe biological system there is a balance between thegeneration of reactive forms of oxygen and their re-

moval by anti-oxidative systems. This state is reflectedby quasi-stationary luminescence at an extremely lowintensity in the wave range of 180–1500 nm. Anydestabilization in this equilibrium (oxidative stress) ismanifested by an increase in UWL. In our study, withthe use of lidocaine or procaine, the intensity of radia-tion increased by several to 15 times, but only after 1/2hours. Lidocaine may act as a scavenger of short-livedfree radicals, including hydroxyl radical. We have ob-served a fall in the intensity of UWL for 634 nm and anincrease for 385 nm. This is an evidence that the anaes-thetic reacts with the reactive forms of oxygen. Wefound also, that the cells showed different sensitivity toanesthetics as well as that the areas of cells locatedcloser to nodes were the most vulnerable.

Our observations by means of spatial-temporal lumi-nescence imaging (Molecular Light Imager) may be avaluable hint in diagnosing the tissue areas more orless vulnerable to the action of local anesthetics, or toother stressful situations.


68 Poster

Protective effect of Aquilegia vulgaris L. extract on aflatoxin B1-induced oxidativestress in rats

Jadwiga Jodynis-Liebert, Marek Murias, Milena Krajewska

Katedra i Zak³ad Toksykologii, Akademia Medyczna im. Karola Marcinkowskiego, ul. Dojazd 30, 60-631 Poznañ

The aim of the study was to investigate the effect ofethyl acetate extract Aquilegia vulgaris L. onmicrosomal lipid peroxidation, glutathione level andantioxidant enzymes activity in the liver of rats intoxi-cated with aflatoxin B1 (AFB1). Animals werepretreated with 12 daily p.o. doses of the extract tested(100 mg/kg b.w.). Then aflatoxin B1 was administeredat a single dose of 1.5 mg/kg b.w. to evoke liver dam-age. �-Tocopherol was used as a positive control.

GSH content was depleted in aflatoxin intoxicatedrats, 1.1 ± 0.1 vs. 5.8 ± 1.1 �mol/g tissue in the controlgroup (p<0.001). The extract prevented the GSH deple-tion (6.5 ± 0.9 �mol/g tissue, p<0.001). Microsomallipid peroxidation stimulated by Fe2+/ascorbate wasenhanced in AFB1-treated group (41.2 ± 3.2 vs. 32.2 ±

4.7 nmol TBARS/mg protein in the control group;p<0.05). The extract caused a decrease of this parame-ter to 29.4 ± 5.2 nmol/mg protein (p<0.05).

The activity of superoxide dismutase, glutathione per-oxidase and catalase were affected neither by AFB1 norAFB1+extract treatment. Glutathione S-transferase ac-tivity, slightly elevated in ABF1-treated rats (2244 ±437 versus 1691 ± 313 U/mg protein in the controlgroup) was reduced in rats pretreated with the extract(1274 ± 302 U/mg protein, p<0.01).

It can be concluded that multiple pretreatment withthe ethyl acetate extract of A. vulgaris protects the liveragainst aflatoxin B1-induced oxidative damage by inhi-bition of lipid peroxidation and preventing reducedglutathione depletion.

69 Poster

Ethanol-, methanol- and ethylene glycol-induced oxidative stress in selected rat or-gans

Agnieszka Jurczyk, Beata Jankowska, Maciej Barzdo, Ewa Meissner, £ukasz Antoszczyk, Jaros³aw Berent,Stefan Szram

Katedra i Zak³ad Medycyny S¹dowej, Uniwersytet Medyczny w £odzi, ul. Sêdziowska 18a, 91-304 £ódŸ

Chronic ethanol ingestion causes toxic effects in or-gans. Extensive studies have shown that ethanol en-hances the production of reactive oxygen species in themicrosomes and mitochondria causing oxidativestress, which results in the development of many alco-hol-related diseases in humans. However, poisoningscaused not only by ethanol, but also by fatal “substi-tutes” of ethyl alcohol are rather frequently encoun-tered in practice in forensic medicine. We investigatedthe influence of three alcohols, viz. ethanol, methanoland ethylene glycol, on oxidative stress parameters inrat organs. We studied changes in the activities ofantioxidative enzymes (catalase and superoxidedismutase) in erythrocytes of inbred male Lewis ratswhich had been administered ethyl alcohol, methyl al-cohol or ethylene glycol for 4, 8 and 12 weeks (Groups I,II and III, respectively). We evaluated oxidative stressparameters in the erythrocytes, livers, kidneys, hearts,lungs and brains of the rats, measuring the concentra-tions of malonyl dialdehyde (MDA), free thiol groups(–SH) and hydrogen peroxide (H2O2). We observedthat ethylene glycol decreased activity of catalase in rat

erythrocytes of Group I (7.5 ± 1.2 U/gHb), Group II (4.6± 1.8 U/gHb) and Group III (5.2 ± 1.2 U/gHb) in com-parison with the control group (9.0 ± 1.1 U/gHb). Activ-ity of superoxide dismutase in erythrocytes of ratswhich were given ethylene glycol changed similarly(2212 ± 175, 2005 ± 107, 1459 ± 220 and 1600 ± 157U/gHb in the control and in Groups I, II and III). Simi-lar decreases were observed in erythrocytes of ratswhich administered other alcohols. The concentrationsof MDA, which is a marker of lipid peroxidation, washigher in the livers of rats which were given ethanol(1.1 ± 0.7, 5.2 ± 1.9; 2.7 ± 1.6 and 1.1 ± 1.0 nmol/mg pro-tein in the control and in Groups I, II and III, respec-tively), and changed similarly in the erythrocytes, kid-neys and brains of the alcohol-consuming rats. We ob-served also significant decrease in the concentration offree thiol groups in the brain (107 ± 35, 19 ± 16, 13 ± 5and 22 ± 10 nmol/mg protein in the control and inGroups I, II and III, respectively) and liver (761 ± 155,358 ± 88, 414 ± 146 and 543 ± 220 nmol/mg protein inthe control and in Groups I, II and III, respectively) inrats which drank ethylene glycol. We observed that eth-


ylene glycol causes also an increase of the concentra-tion of hydrogen peroxide in all investigated organs.Our results suggest that ethanol, methanol and ethyl-ene glycol generate oxidative stress in rat organs and

may change activities of antioxidative enzymes, causelipid peroxidation and protein damage, and increasehydrogen peroxide production.

70 Poster

Protective effect of inosine against oxidative-stress induced DNA damage

Beata Kaliñska-B³ach, Aleksandra Gil, Halina Ha³oñ, Micha³ WoŸniak

Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk

Oxygen derived free radicals are known to inducestrand breaks or base modifications in DNA. DNAstrand breaks are often observed in many pathologiesand apoptosis as well. We have examined the ability ofinosine and 2’-deoxyinosine to protect pBR322supercoiled plasmid DNA from the Fe-dependentstrand breakage induced by reactive oxygen species.

DNA strand breakage was measured by the conver-sion of supercoiled pBR 322 double-stranded DNA toopen circular or linear form. There three forms can beseparated by agarose gel electrophoresis, because theirelectrophoretic mobilities are different.

Supercoiled plasmid DNA (100 ng in 50 mM sodiumphosphate pH 7.4) was incubated at room temperaturefor 1 h with 100 �M H2O2 and different concentrationsof iron ions (1–5 �M Fe2+) with or without inosine and2’-deoxyinosine (0.1–2 mM). After incubation the sam-ples were separated by electrophoresis in a 1% agarosegel stained with ethidium bromide.

In biological systems high reactive hydroxyl radicalsare produced mainly in the Fenton reaction, where irontakes part. However, activity of other oxidizing speciessuch as the ferryl ion can not be excluded. It has beenfound that ROS converted supercoiled plasmid DNA toopen circular or linear form in a Fe-dependent manner.The extent of DNA scission is evident by comparing un-treated DNA (control) with treated DNA and the levelof degradation depends on the concentration of ironions (50 or even 100% degradation).

The aim of present experiments was to evaluate theprotective effect of inosine on iron dependent DNA deg-radation. Inosine and 2’-deoxyinosine were found toprevent DNA against this oxidative damage. We sug-gest that iron binding and delocalization can be respon-sible for antioxidative properties of inosine. Takinginto consideration that purine ring of inosine containsa hydroxyl group, there is a possibility of binding anddelocalization of Fe2+ by this nucleoside.

71 Poster

Activity of superoxide dismutase and catalase in erythrocytes of workers occupation-ally exposed to lead

S³awomir Kasperczyk1, Aleksandra Kasperczyk1, Ewa Birkner1, Jolanta Zalejska-Fiolka1, Maria Dziwisz2

1 — Katedra i Zak³ad Biochemii Ogólnej w Zabrzu, Œl¹ska Akademia Medyczna w Katowicach, ul. Jordana 19, 41-808 Zabrze, 2— NZOZ Eko-Prof-Med, Centrum Medyczne, Miasteczko Œl¹skie

Lead is one of the common toxic metals present in ourenvironment. It has been found that lead negatively in-fluences the circulation system, and most often it hasbeen connected with oxidative stress and lipid peroxi-dation and can also modify activity of antioxidative en-zymes, but informations were often contradictory.

The population studied included healthy workersfrom smelting works of zinc and lead. Blood leadlevel (PbB) and concentration of zinc protoporphyrin(ZPP) were employed to evaluate the degree of expo-sure. Control group (n=35) was administration work-ers with normal levels of PbB and ZPP in blood.

Workers occupationally exposed to lead were dividedinto two subgroups: low-lead exposed (LL) (n=75)with PbB=25–40 mg/dl and high-lead exposed (HL)(n=62) with PbB=40–61 mg/dl. Activities of super-oxide dimutase (SOD) and catalase (CAT) were deter-mined in erythrocytes.

Data are shown as mean ± SEM. Activity of SOD was19.9 ± 1.19 NU/mg Hb in HL group, 18.8 ± 0.99 NU/mgHb in LL group and 16.1 ± 1.28 NU/mg Hb in control.Activity of CAT was 264 ± 8.01 IU/mg Hb in HL group,265 ± 6.85 IU/mg Hb in LL group and 254 ± 7.61 IU/mgHb in control. It was found that the activity of SOD in-


creases in both lead exposed groups (p=0.049 LL groupin comparison to control and p=0.021 HL group in com-parison to control). There were no changes of the CATactivity in the population studied. There was also posi-

tive correlation between PbB and SOD activity (R=0.18p=0.022).

The increase of SOD activity in erythrocytes seems tobe an adaptive mechanism to lead toxicity.

72 Poster

Protein oxidation and rapid induction of megamitochondria formation indoxorubicin-treated cardiomyocytes

Jakub Kêdzior1, Dorota Kulawiak-Ga³¹ska1, Jan Spodnik2, Micha³ WoŸniak1, Takashi Wakabayashi3

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Katedra Anatomiii Neurobiologii, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 3 — Department of Cell Biology and MolecularPathology, Medical University of Gdañsk, ul. Dêbinki 1, 80-211 Gdañsk

Mitochondria undergo several structural changeswhich are an outcome of functional changes occurringboth in physiological and pathological conditions. For-mation of megamitochondria (MG) occurs in many dis-eases i.e. ethanol intoxication, mitochondrial myo-pathy or muscle dystrophy. Three mechanisms of theformation of MG are considered:

1) Inhibition of mitochondrial proteins synthesis, 2)Suppression of division of mitochondria, 3) Fusion ofmitochondria.

In the present study we observed rapid changes inmorphology of mitochondria after administration of ananticancer drug, doxorubicin (DOX; 0.2–10 �M). Firstchanges were clearly seen already after 10 minutes.Fully formed MGs were observed after 1 h. It is theshortest time of inducing MG with chemicals known in

the literature. The obtained data are a strong indirectevidence supporting the theory of MG formation by fu-sion of mitochondria.

Moreover, the level of protein and lipid peroxidationin the mitochondrial fraction from the Syrian hamsterheart treated with DOX (5 mg/kg of body weight), wasmeasured by evaluating MDA and protein carbonylgroups (CO). The level of protein oxidation was ele-vated (1.02 vs. 0.32 nmol CO/mg protein 10 min afterDOX administration vs. control) while there was nochange in the level of lipid peroxidation (0.1 nmolMDA/mg protein, both in the control and 10 min afterDOX administration). If similar effects appear under invitro conditions, it will mean that that protein oxidationis an event preceding lipid peroxidation in the processof megamitochondria formation.

73 Poster

The influence of metals administered in diet on the level of chosen elements of anti-oxidant barrier in rats

Ma³gorzata Kie³czykowska, Kazimierz Pasternak, Irena Musik, Jolanta Wroñska

Katedra i Zak³ad Chemii Ogólnej, Akademia Medyczna w Lublinie, ul. Staszica 4, 20-081 Lublin

Lithium is used in psychiatry for the treatment ofmanic and depressive illness and for thepotentialization of the action of other drugs. Its admin-istration can decrease negative side effects in patientsreceiving chemotherapy for leukaemia. Aluminium ex-erts neurotoxic, lithium teratogenic, lead and chro-mium carcinogenic influence. The importance of thecorrect action of the antioxidant barrier made us inves-tigate the effect of above-mentioned metals administra-tion on glutathione peroxidase (GPx) and superoxidedismutase (SOD) activities in rat’s serum and tissues.

Our study was carried on male Wistar rats. The ani-mals were divided into five groups: I — control; II, III,

IV — received water solutions of Al2(SO4)3 � 18H2O,Pb(NO3)2 and Cr(NO3)3 � 9H2O respectively, in theform of drinking water at the dose of 500 mg of metal �dm–3; V — received the water solution of Li2SO4 � H2Oin the form of drinking water at the dose of 150 mg Li �dm–3. A half of animals of each group were killed afterthree weeks and the rest after another three weeks. Therats were sacrificed under ketamine narcosis and bloodfrom the heart as well as tissues of liver, kidney, brain,spleen, femoral muscle and heart muscle were col-lected. Serum was separated, 10% (w/v) tissuehomogenates were prepared in 0.1 mol/L Tris/HClbuffer pH = 7.4. In serum and supernatants glutathione


peroxidase and superoxide dismutase activities weremeasured using RANSOD and RANSEL kits producedby RANDOX.

After three weeks of intoxication the activities of bothenzymes in serum were decreased except SOD in lith-ium receiving group whereas after six weeks GPx activ-ity increased and SOD activity decreased in all groups.The administration of discussed metals for the periodof three weeks caused the increase of GPx and SOD ac-tivities in heart muscle and liver and their decrease inbrain. After six weeks GPx activity decreased in spleen

and liver and enhanced in heart muscle and kidney.The changes of the tissue enzymes activities during allthe experiment were mostly univocal in the case of lith-ium. The influence of the other metals depended ontime of intoxication and tissue. In many cases when theactivity of one of discussed enzymes decreased the ac-tivity of another one enhanced.

The obtained results indicate that administration ofchromium, lead, aluminium and lithium disturbs theaction of antioxidants.

74 Poster

Early antioxidative response in hormonally induced experimental carcinogenesis

Jaros³aw Kobiela, Jaros³aw Meyer-Szary, Paulina Kryger


Recent epidemiological studies report increasingbreast cancer incidence. One of the important risk fac-tors is the increased serum level of estradiol due to oralcontraceptives, delayed first pregnancy, reduced over-all number of pregnancies in life, hormonal substitutivetherapy and elevated food content of this hormone. Inthis study we used an experimental model of hor-monal-dependent nephrocarcinogenesis. The pathome-chanism of this cancer induction is related to estradiol4-hydroxylation and redox cycling of its quinone andsemiqinones, with generation of free radicals. It is wellknown that severe oxidative stress can lead to apoptosisor even necrosis, which is not observed in this model.The non-specific enzymatic antioxidative response canplay an important part in the initial stage of hormoneexposure. One of the enzymes suspected for oxidativedamage repair is (oxidative-stress inducted) micro-somal glutathione-S-transferase (mGST). Aim of thestudy: Detection of oxidative stress at the initial stage ofestrogen-induced carcinogenesis and evaluation of pos-sible physiological enzymatic mechanisms protectingthe kidney tissue.

Material and methods: 90 male Syrian hamsters were di-vided in 3 groups: Group 1 was injected i.p. with 70 mg/kgb.w. of estradiol. Animals of subgroups 1a, 1b, 1c were sacri-ficed after 1, 3, 5 h, respectively, and their kidneys were ex-cised for further examinations. Animals of Group 2 were s.c.implanted with 250 mg/kg b.w. of estradiol and sacrificed af-ter 30 days of hormone administration. Group 3 did not re-ceive any hormone and served as a control. The excised or-gans were macro- and microscopically investigated and bio-chemical analyses were performed. The homogenates werefractionated by differential centrifugation. To assess the oxi-dative damage of cellular biomolecules, the levels of carbonyland sulphydryl groups, malondialdehyde, hydroperoxidesand 8-oxo-dG were measured. The activities of cGST andmGST were also assessed.

Results: Redox cycling of estradiol derivatives leads toselective peroxidative damage at the level of proteins (car-bonyl groups : 1.8, 4.4, 9.1, 2.4 and 3.8 nmol/mg protein inthe control and after 1, 3 and 5 h, and 1 month, respec-tively), but not lipids. An induction of mGST activity wasobserved (4.53, 6.97, 6.46, 9.40 and 3.15 U/ml in the con-trol and after 1, 3 and 5 h, and 1 month, respectively).

75 Poster

Protein oxidation and histopathology in experimental nephrocarcinogenesis

Jaros³aw Kobiela1, Jacek Krajewski1, Tomasz Stefaniak2

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — II KlinikaChirurgii Ogólnej, Gastroenterologicznej i Endokrynologicznej, Akademia Medyczna w Gdañsku, Gdañsk

Introduction: Estradiol-induced model of nephrocarci-nogenesis is one of the most commonly used models of ex-perimental hormone-dependent carcinogenesis. Although

it is thought to mimic human breast cancer (estradiol-4-hy-droxylase), its histogenesis still remains unclear. Oberleysuggests interstitial cells as a possible origin of the neo-


plasm, while Goldfarb supports the hypothesis of epithe-lial cells as origin cells. Although estradiol receptors werefound in interstitial cells, the exclusive contribution ofthese cells was not definitely confirmed.

The aim of this study is to present morphological andmicroscopic observations in relation to biochemicalanalysis and current opinions. Moreover, due tolong-lasting experience in these models, we would liketo present primary observed by our team extrarenalchanges i.e. involving liver (liver cysts, cholecistol-ithisis), skin (retention type pigmentation aberrations)and skeletal system).

Material and methods: 40 male Syrian hamsters weres.c implanted with 250 mg/kg b.w. of estradiol. Animals

from each group were sacrificed after 1, 3, 6, 9 and 12months. Another 15 animals did not receive any hor-mone and were sacrified as a control in time-matched in-tervals. Part of the kidney tissue was sectioned andstained with eosine and hematoxyline. The remainingpart was homogenized and peroxidative protein damagewas assessed. Results: Oxidative damage revealed in thetissue correlates with the intensity of microscopic and,later, macroscopic changes. Severity of microscopicchanges is gradually increasing from 3 months, whilesome diffused, preneoplastic changes are observed assoon as after one month. After the period of 9 and 12months the incidence of the neoplasm is close to 100%with appearance of additional extrarenal changes.

76 Poster

Interaction of nitroxides and mitoxantrone with the plasma membrane ofcardiomyocytes

Aneta Koceva-Chy³a1, Adam Sokal2, Katarzyna Kania1, Krzysztof GwoŸdziñski3, Zofia JóŸwiak1

1 — Department of Thermobiology, University of £ódŸ, ul. Banacha 12/16, 90-237 £ódŸ, 2 — I Clinical Department of Cardiology,Silesian Centre of Heart Diseases, Zabrze, 3 — Department of Molecular Biophysics, University of £ódŸ, £ódŸ

Cardiotoxicity of anticancer drugs depends mainly onoxygen free radical-induced oxidative stress, generatedduring their metabolic activation inside the cell.Cardiomyocytes are relatively unprotected against oxi-dative stress due to the low level of the intracellular an-tioxidant enzymes. Therefore one of the last trends incontemporary chemotherapy is the search for non-toxiccompounds of low molecular weight that applied incombination with the anticancer drugs could lessentheir cardiotoxic effects. Nitroxides, cell permeable sta-ble radicals with antioxidant activity were shown toscavenge effectively superoxide anions by mimickingsuperoxide dismutase activity and to protect cells fromlipid peroxidation and cytotoxicity of hydrogen perox-ide and superoxide.

This work was aimed at estimation of the protective ef-fects of pyrroline and pyrrolidine nitroxides (Pirolin andPirolid) on the plasma membrane of cardiomyocytestreated with the anticancer drug mitoxantrone (MTX).

Myocytes were isolated from hearts of healthy maleWistar rats and incubated at 37oC with different concen-trations of MTX, nitroxides or combination of both.Plasma membrane fluidity was estimated on the basis offluorescence anisotropy of the hydrophobic fluorescentprobe 12-AS. We found that MTX at concentrationsabove 1 �M caused a significant dose-dependent de-crease in fluidity of the hydrophobic core of lipid bilayer.Pirolid by itself induced changes in the structure of theplasma membrane and increased its fluidity at concen-trations higher than 50 �M. Pirolin alone had little effecton membrane fluidity. Preincubation of cardiomyocyteswith low concentrations of Pirolin (<100 �M) attenuatedonly partly the toxic effect of MTX as the plasma mem-brane of cardiomyocytes treated with the combination ofboth compounds (a drug and a nitroxide) was still morerigid compared to control (untreated cells). Little protec-tive effect was observed for the lower concentrations(20–100 �M) of Pirolid.

77 Poster

Peroxynitrite and fibrinolytic system; the effect of peroxynitrite on plasmin activity

Joanna Ko³odziejczyk, Pawe³ Nowak, Fadwa Majeed, Barbara Wachowicz

Katedra Biochemii Ogólnej, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ

Peroxynitrite (ONOO–) is a powerful, highly reactiveand a relatively long-lived cytotoxic oxidant formed invivo in the rapid reaction of nitric oxide and superoxide

anion. In biological systems, peroxynitrite or its reac-tive intermediates can attack a wide variety of biologi-cal molecules, including proteins, lipids, carbohydrates,


and nucleic acids. It has been demonstrated that reac-tion of protein with ONOO– results in the oxidation oftryptophan, cysteine and methionine, tyrosine nitra-tion, dityrosine formation, carbonyl formation and pro-tein fragmentation. Previous studies suggested thatamong plasma proteins plasminogen might be a poten-tial target for peroxynitrite-mediated nitration of pro-tein in plasma, therefore we investigated the effects ofperoxynitrite on function of plasminogen.

We showed that peroxynitrite modified plasminogen ina concentration-dependent manner, resulting in the inhi-bition of both plasmin amidolytic activity and conversion

of plasminogen to plasmin. The effects of peroxynitrite ac-tion were partially inhibited by antioxidants (gluthatione,deferoxamine, uric acid). SDS/PAGE analysis of peroxy-nitrite-treated plasminogen showed some additionalbands on the top of the gel (aggregated material) and thepresence of nitrotyrosine. The amount of nitrotyrosine,detected by immunoassay with monoclonal anti-nitro-tyrosine antibodies, was peroxynitrite concentra-tion-dependent. Our results suggest that peroxynitritemight play a role in modulation of fibrinolytic activity.This research was supported by a grant from University of

Lodz (505/426).

78 Poster

Estimation of the genotoxic properties of novel class of advanced glycation end prod-ucts (AGE) by measuring micronuclei in human lymphocytes in vitro

Maria Konopacka1, Jacek Rogoliñski1, Zbigniew Maniakowski2, Magdalena Staniszewska3, Ewa Sowula3,Danuta Witkowska3, Andrzej Gamian3

1 — Zak³ad Radiobiologii Doœwiadczalnej i Klinicznej, Centrum Onkologii w Gliwicach, ul. Armii Krajowej 15, 44-100 Gliwice,2 — Zak³ad Fizyki Medycznej, Centrum Onkologii w Gliwicach, ul. Armii Krajowej 15, 44-100 Gliwice, 3 — Instytut Immunologiii Terapii Doœwiadczalnej, Polska Akademia Nauk, ul. Rudolfa Weigla 12, 53-114 Wroc³aw

It was previously observed in colorectal carcinomacells, that there is association between the cancer inva-sive activities of cells and their exposure to advancedglycation end products (AGE) [1]. Incubation of pig kid-ney cells with AGE-BSA (AGE-bovine serum albumin),carboxymethyllysine-BSA as well as methylglyoxal-BSA resulted in a significant increase in DNA damageand this effect was mediated by RAGE [2]. We exam-ined the novel class of high molecular AGEs formedfrom protein and oligosaccharide as well as low molecu-lar mass AGEs obtained from the oligosaccharide andN-acetyllysine and N-acetylarginine. The obtainedproducts possessed different epitopes than productssynthesized from commonly used carbohydrates or car-

bohydrates degradation products. We have observedby measuring of micronuclei in human lymphocytes invitro that the novel high molecular mass glycation endproducts expressed the genotoxicity. The number ofmicronuclei in human lymphocytes reached 40.22 ±5.34 promille (MN/1000CBL), comparing to 28.80 ±6.50 MN/1000CBL for the reference methylglyo-xal-BSA, whereas a control value was 20.66 ± 1.39MN/1000CBL. The low molecular mass glycation prod-ucts did not exhibit genotoxicity.References:1. Kuniyasu H, Chihara Y, Kondo H (2003) Int J Cancer, 104:

722–727.2. Stopper H, Schinzel R, Sebekova K, Heidland A (2003) Can-

cer Lett, 190: 151–156.

79 Poster

Effect of quinapril and perindopril therapies on the glutathione concentration andthe glutathione peroxidase and glutathione-S-transferase activities in aged patientswith arterial hypertension (a preliminary study)

Katarzyna Kot³owska1, Karolina Szewczyk-Golec1, Jolanta Czuczejko1, Hanna Pawluk1, KorneliaKêdziora-Kornatowska2, Jadwiga Motyl2, Józef Kêdziora1

1 — Katedra i Zak³ad Biochemii, Akademia Medyczna im. Ludwika Rydygiera, ul. Kar³owicza 24, 85-092 Bydgoszcz, 2 — Katedrai Klinika Geriatrii, Akademia Medyczna im. Ludwika Rydygiera, ul. M. Sk³odowskiej-Curie 9, 85-094 Bydgoszcz

Reactive oxygen species (ROS) are suggested to be in-volved in the pathogenesis of arterial hypertension.

During aging ROS concentrations increase both in thecells and in plasma. Organisms have developed the


antioxidative system that prevents or neutralizes thedeleterious ROS effects, but the capabilities ofantioxidative mechanisms evidently decrease with ad-vanced age. Glutathione (GSH), glutathione pero-xidases (GSH-Px) and glutathione-S-transferases(GST), which play an important role in the pro- andantioxidative mechanisms, are markers of these pro-cesses.

The aim of our study was the determination of the ef-fect of quinapril and perindopril (the angiotensin con-verting enzyme (ACE) inhibitors) on the level of GSHand the GSH-Px and GST activities.

The blood was taken from aged patiens with essentialarterial hypertension (mean age 74 years) beforequinapril and perindopril treatment (day “0") and after7th (day ”7") and 45th days (day “45") from the begin-

ning of quinapril and perindopril therapies. The GSHconcentration in whole blood was assayed by themethod of Beutler. The GSH-Px activity was deter-mined in haemolysates according to Paglia and Valen-tine with t-butylhydroperoxide as substrate, the GSTactivity according to Habig et al. using 1-chloro-2,4-di-nitrobenzene (CDNB) as a substrate. The haemoglobinconcentrations in haemolysates were estimated by theDrabkin method.

The GSH concentration and the GSH-Px and GST ac-tivities did not differ statistically significantly duringthe treatment and compared with “0" day.

Our investigation indicate that quinapril andperindopril do not affect on the GSH concentration andthe GSH-Px and GST activities in geriatric patients suf-fering with arterial hypertension.

80 Poster

Selected cardiological drugs and oxidative status

Edward Kowalczyk1, Maria Kopff2, Anna Kopff3

1 — Zak³ad Biofizyki i Fizjologii Cz³owieka Wydzia³ Wojskowo-Lekarski, Uniwersytet Medyczny w £odzi, Pl. Hallera 1, 90-647£ódŸ, 2 — Zak³ad Chemii i Biochemii Klinicznej Wydzia³ Fizjoterapii, Uniwersytet Medyczny w £odzi, Pl. Hallera 1, 90-647 £ódŸ,3 — Klinika Kardiologii IMW, Uniwersytet Medyczny w £odzi, £ódŸ

Monovalent and/or divalent reduction of molecularoxygen leads to the production of partially reducedforms of oxygen (PRFO) which include active oxygen,superoxide anion, hydrogen peroxide and hydroxyl rad-ical. These PRFO are highly reactive and have been im-plicated in the pathogenesis of a variety of cardiac dis-eases including ischemia-reperfusion injury, catechol-amine-induced arhythmias and drug-inducedcardiotoxity. Lipid peroxidation products as well as oxi-dation of functional proteins have been suggested to af-fect cardiac and endothelial cells function.

The aim of this study was to determine the effect ofdrugs frequently applied in cardio-vascular diseases(metoprolol, acetylsalicylic acid, simvastatin andmolsidomine) on the antioxidantive/oxidantive balance.The study was performed on 40 healthy rabbits. The oxi-dative status was determined basing on the measure-ments of: (1) thiobarbituric acid reactive species (TBARS— lipid peroxidation products; the Rice-Evans method), (2)protein carbonyls groups (marker of proteins oxidative in-jury; the methods of Levin et al. and Reznik), (3)nitrotyrosine (marker of NO-mediated tissue damage; themethods of Khan et al. and Ter Steeg et al.) and (4)sulfhydryl groups concentration (protein oxidation prod-uct; the Ellman method).

The assays were performed in the plasma and wholeblood of rabbits after three weeks of daily intragastric ad-ministration of metoprolol (6.42 mg/kg of body mass),acetylsalicylic acid (4.29 mg/kg of body mass),simvastatin (3.43 mg/kg of body mass) and molsidomine(0.69 mg/kg of body mass). At the same time the controlgroup of rabbits was given water by gastric tube.

We found that all drugs except acetylsalicylic acidcaused an increase in the plasma and hemolysate levels ofTBARS (plasma TBARS: control, 0.78 ± 0.14 �M;metoprolol, 1.06 ± 0.19 �M, p<0.01; simvastatin, 0.95 ±0.34 �M, p<0.05; molsidomine, 1.35 ± 0.28 �M, p 0.05;TBARS concentration in the hemolysates: control, 1.80 ±0.15 �M, metoprolol, 3.25 ± 0.18 �M, p<0.001;simvastatin, 2.40 ± 0.39 �M, p<0.05; molsidomine, 2.51 ±0.14 �M, p 0.05). No changes in nitrotyrosine concentra-tion were observed after drug administration. The con-tent of carbonyl groups did not change after metoprolol,but increased significantly after simvastatin andmolsidomine application. Blood sulfhydryl group concen-tration did not change after metoprolol but decreased sig-nificantly after acetylsalicylic acid and increased signifi-cantly after molsidomine administration.The study was financed by the Medical University of £ódŸ

through the intramural grant No.502-17-018.


81 Poster

Cu(II)-catalyzed oxidation in the presence of hydrogen peroxide of the 1-10, 1-16fragments of human and mouse beta-amyloid peptide

Teresa Kowalik-Jankowska1, Monika Ruta1, Kornelia Wiœniewska2, Leszek £ankiewicz2

1 — Wydzia³ Chemii, Uniwersytet Wroc³awski, ul. Joliot Curie 14, 50-383 Wroc³aw, 2 — Wydzia³ Chemii, Uniwersytet Gdañski,ul. Sobiewskiego 18, 80-952 Gdañsk

The oxidative modification of proteins by reactiveoxygen species (ROS) is implicated in the etiology orprogression of a number physiological disorders anddiseases. The involvement of ROS in Alzheimer’s dis-ease (AD) is indicated by the observation that the lev-els of nitrotyrosine, protein carbonyl and malondi-aldehyde are elevated in patients with AD. The ROSmay be formed by any one of a large number of physio-logical and nonphysiological processes. Among othersthese include the formation of hydrogen peroxide byendogenous oxidases and the conversion of H2O2 tohydroxyl radical by metal-catalyzed oxidation (MCO)systems. The large body of evidence indicates that thehomeostases of the red-ox active transition metals(Fe2+, Cu2+) are significantly altered in the AD brainand that these metals are present in the neuropatho-logy.

In an attempt to elucidate the products of themetal-catalyzed oxidation of the human (H) and mouse(M) (1-10H), (1-10M), (1-16H) and (1-16M) fragments of�-amyloid peptide, the high performance liquid chroma-tography (HPLC) and matrix-assisted laser desor-ption/ionization mass spectrometry (MALDI-MS) meth-ods and Cu(II)/H2O2 as a model oxidizing system wereemployed.

Oxidation targets for all peptide studied are thehistidine residues coordinated to the metal ions. Forthe (1-16H) peptide are likely His13 and/or His14, andfor the (1-16M) fragment His6 and/or His14, which areconverted to 2-oxo-His. Metal binding residue, the as-partic acid (D1) undergoes the oxidative decarbo-xylation and deamination to pyruvate. The cleavages ofthe peptide bonds by either the diamide or �-amidationpathways were also observed.This work was supported by the State Committee for Scien-

tific Research (KBN 3 T09A 069 18).

82 Poster

Attenuated kallikrein-mediated kinin release from kininogens oxidized bymyeloperoxidase — hydrogen peroxide — chloride system

Andrzej Kozik, Katarzyna Jekiel, Ibeth Guevara-Lora, Maria R¹pa³a-Kozik

Zak³ad Biochemii Analitycznej, Wydzia³ Biotechnologii, Uniwersytet Jagielloñski, ul. Gronostajowa 7, 30-387 Kraków

Bradykinin and related oligopeptides, collectivelycalled kinins, are universal mediators of inflammationthat are produced by the proteolytic action of kallikreinenzymes on precursor proteins, kininogens. Possiblemodifications of the components of kinin-formationsystem by numerous factors operating during inflam-matory episodes are poorly recognized. For example,the multifunctional kininogen proteins may be alteredsignificantly by reactive oxygen species that are re-leased at the inflammatory foci during phagocytosis ofpathogen particles by recruited neutrophils. One typeof these aggressive oxidants is hypochlorous acid thatis produced from hydrogen peroxide and chloride ionsby myeloperoxidase, the enzyme released fromneutrophil granules upon activation of these cells.

The aim of this work was to oxidize kininogen mole-cules with myeloperoxidase — hydrogen peroxide —

chloride system and to determine the effect of this mod-ification on three functionally essential properties ofkininogens, i.e. (1) kallikrein-mediated kinin release,(2) inhibitory activity of kininogens against cysteineproteinases, e.g. papain, (3) interaction of high-mole-cular mass kininogen with prekallikrein, a necessaryprerequisite of intrinsic blood coagulation pathway.

Amino acid analysis of oxidized kininogens showednearly complete conversion of methionine residues tomethionine sulfoxide residues. Other amino-acid resi-dues were insignificantly altered. Although somemethionine residues are present both in the cysteineproteinase inhibitor domains and in the prekalli-krein-binding domain of kininogen molecule, neither ofthese functional properties were significantly differentin oxidized kininogens from those in the native proteins.However, the oxidized kininogens, both high molecular


mass- and low molecular mass forms, are poorly suscep-tible to the processing by plasma- and tissue kallikreins,respectively. Hence, the kinin release from kininogenswas selectively attenuated after oxidation of thesekinin-precursor molecules. The attenuation effect wasdramatic in the high molecular mass kininogen —plasma kallikrein system and moderate in the tis-

sue-kallikrein dependent kinin-formation systems.These results suggest that the oxidation of kininogens,specifically of the methionine adjacent to the internalkinin sequence, is likely to occur at the inflammatorysites and to quench the kinin-forming cascade at the latestage of inflammation.

83 Poster

Oxidative stress participates in the response of yeast cells to hyperosmotic challenge

Sabina Kozio³, Ewa ¯yracka, Tomasz Biliñski, Grzegorz Bartosz

Instytut Biologii i Ochrony Œrodowiska, Uniwersytet Rzeszowski, ul. Rejtana 16C, 35-310 Rzeszów

Similarity of the response of yeast Saccharomycescerevisiae to oxidative stress and other types of stressincluding osmotic, heat and ethanol stress has beennoted mainly on the basis of cross-tolerance and geneinduction pattern. It has been reported recently(Garay-Arroyo et al., 2003) that yeast devoid ofCuZnSOD is hypersensitive to hyperosmotic stress. Wefound that the rate of production of reactive oxygenspecies (estimated fluorimetrically by oxidation of2’,7’-dichlorofluorescin) by yeast is several-fold higherin hyperosmotic medium (containing 0.8 M NaCl) thanin control medium. This effect is accompanied by a pro-

gressive loss of acid-soluble thiols and of total antioxi-dant capacity of the acid-soluble fraction of cells over3-h incubation in the hyperosmotic milieu. Antioxi-dants (10–30 mM ascorbate, 0.05–1 mM glutathione,0.5–1 mM cysteine and 0.1–1 mM dithiothreitol) pro-tect CuZnSOD-deficient strains allowing them to growin hyperosmotic medium (0.8 M NaCl). Anaerobic con-ditions also ameliorate growth restriction induced byhyperosmotic medium. These findings suggest that oxi-dative stress is imposed in yeast cells by hypertonic me-dium and participates in the growth restriction inducedby hypertonic stress.

84 Poster

Free radicals scavenging by amine N-oxides

Anna Krasowska1, Anna Mróz1, Jacek £uczyñski2, Andrzej Piasecki2

1 — Instytut Genetyki i Mikrobiologii, Uniwersytet Wroc³awski, ul. Przybyszewskiego 63-77, 50-148 Wroc³aw, 2 — Instytut ChemiiOrganicznej i Tworzyw Sztucznych, Politechnika Wroc³awska, Wroc³aw

Our aim was the synthesis of derivatives of alkyl-amidoamines and alkylaminoesters containing tertiaryamine N-oxide moiety with antioxidative activity andexplanation the mechanism of action of these sub-stances.

The properties of the above mentioned derivativesmay be easily changed (hydrophilic-lipophilic balanceinfluences substantially the incorporation of the mole-cules into cell membrane) by the change of alkyl radicallength, distance between amide and N-oxide groupings,and number of polar (amide and N-oxide) groupings.

The new group of antioxidants shows specificityagainst certain types of free radicals. Contrary to

N-oxides of alkylaminoesters, derivatives of alkyla-midoamines are capable to neutralising peroxyl radi-cals in chemiluminescence test, in in vitro tests of con-jugated dienes, and TBRS measurement (IC50 40–70�M). The activity of N-oxides was dependent on thelength of alkyl chain and homologues with shorterchain than of 13 carbon atoms were inactive.

Some of N-oxides stimulated the growth ofSaccharomyces cerevisiae �sod mutants suggesting theprotective properties against superoxide radical in thecells.

These findings constitute a promising basis for the in-tensive future research.


85 Poster

Effect of �-amyloid and N-lauroylethanolamine on rat heart mitochondrialmembranes

Dorota Kulawiak-Ga³¹ska1, Olivia Zacharek1, Micha³ WoŸniak1, Jerzy Nowak2

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Katedra i Zak³adBiofizyki, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk

�-Amyloid peptide is a constituent of neuronal andnon-neuronal cells. It accumulates in the extracellularspace of neuronal cells and plays an important role inthe pathogenesis of Alzheimer’s disease but the mecha-nism of its toxicity is not fully understood.

�-Amyloid can also accumulate in the cells of varioustissues, especially in the kidney, spleen, liver and heart.

The peptide is also able to insert into the lipid bilayer.The insertion is controlled by the ratio of cholesterol tophospholipids and, itself, can affect fluidity of the lipidbilayer.

N-Acylethanolamines are generated during heartischaemia and can be engaged in the tissue protection.

The aims of the study were: to investigate the effect of�-amyloid peptide on the fluidity of mitochondrialmembranes and the protective effect of N-lauroyl-

ethanolamine on the disturbance of membrane fluidityinduced by �-amyloid.

Mitochondria were obtained from rat heart. Amyloid�-protein fragment 25–35 was used. Changes of mem-brane fluidity were measured by the spin-labeling tech-nique using 5-doxyl-stearic acid. The ratio of the heightof the central line to the height of the highfield line ofthe electron spin resonance spectra was analysed as ameasure of membrane fluditity.

Exposure of mitochondria to the �-amyloid peptidefragment studied results in a significant increase of mi-tochondrial membrane fluidity.

N-Lauroylethanolamine displays a potential protec-tive effect against the perturbation of membrane fluid-ity induced by the �-amyloid peptide.

86 Poster

Regulated overexpression of mitochondrial superoxide dismutase (MnSOD) in10T1/2 cell line

Tomasz Loch, Eva Bober, Thomas Braun

Institut für Physiologische Chemie, Martin Luther Universität Halle-Wittenberg, Hollystr. 1, Halle, Germany

Vast majority of oxygen radicals is produced in themitochondrial electron transport chain. Manganesesuperoxide dismutase (MnSOD, SOD2) catalyzes theconversion of superoxide anion to hydrogen peroxideand oxygen. Expression of the MnSOD gene is respon-sive to external and internal stimuli while protein activ-ity changes in different cell cycle phases being the low-est in the S-phase and the highest in G0. It has beenshown that cells overexpressing MnSOD are more re-sistant to apoptosis and have diminished growth rate.Overexpression of MnSOD in many cancer cell linessuppresses their tumorigenecity.

We have stably transfected 10T1/2 fibroblasts with aconstruct coding for murine MnSOD under the controlof CMV minimal promoter driven by tetracycline re-sponsive element. It also produces green fluorescenceprotein as a reporter molecule. Regulation of the ex-pression of this construct is achieved by tetracy-cline-controlled transactivator which was also intro-duced into these cells. A clone with the lowest back-ground and the highest level of inducibility was chosenfor further studies. In addition, another stable cell linewas established with a construct employing siRNA todownregulate native MnSOD expression.


87 Poster

Comparison of DNA damage and repair kinetics in lymphocytes of cancer patientsand healthy donors

Bo¿ena Lubecka1, Maria Wide³1, Zofia Ko³osza2, Sylwia Jêdruœ3, Joanna Rzeszowska1

1 — Zak³ad Radiobiologii Doœwiadczalnej i Klinicznej, Centrum Onkologii Oddzia³ Gliwice, Wybrze¿e Armii Krajowej 15, 44-101Gliwice, 2 — Zak³ad Epidemiologii Nowotworów, Centrum Onkologii Oddzia³ Gliwice, Gliwice, 3 — Klinika GinekologiiOnkologicznej, Centrum Onkologii Oddzia³ Gliwice, Gliwice

Correct repair of DNA damage plays a protective roleagainst genotoxic factors, while its impairment leads tomutation and may be a basis of cancerogenesis. Singlecell gel electrophoresis (comet assay) is a sensitive andeasy method for evaluation of DNA damage and kinet-ics of its repair and is widely used in environmental mu-tagenesis and radiobiological studies.

In this study the alkaline version of the comet assaywas applied for comparison of radiation-induced DNAdamage and repair capacity in peripheral blood lym-phocytes of 42 patients with cervical carcinoma and 30healthy women.

The results indicate a great variation in response toradiation in both groups under the study. However, thelymphocytes of patients were characterized by a signifi-cantly higher level of background damage(p<0.000005), increased sensitivity to radiation(p=0.002) and reduced DNA repair potential (p=0.001).

Furthermore, these features were more pronounced forpatients with familial anamnesis. The influence ofgamma irradiation was additionally analyzed with re-spect to age and smoking status. The influence of ageon repair kinetics was significant in healthy women. Inthe group of patients the oldest patients repaired DNAdamage more slowly than younger patients but the dif-ferences were not significant. The smoking status in-creased the level of basal damage (p<0.03) and de-creased lymphocyte repair ability (p<0.005) in healthydonors. The relationship between smoking and repairimpairment was more complicated for cancer patients.

Analysis of results for donors indicates that lympho-cyte comet assay can be useful for estimation of influ-ence of genotoxic factors. The data of comet assay in can-cer patients could suggest that impairment of lympho-cyte response is a predisposing factor to cervix cancer.Partly supported by KBN grant No. 4P05A.015.19

88 Poster

High iodine doses induce oxidative stress and decreases functional activity of ratthyroid cells

Siarhei Lupachyk, Zoya Niatsetskaya, Liliya Nadolnik

Laboratory of Endocrine Glands Biochemistry, Institute of Biochemistry of NAS, BLK-50, 230017 Grodno, Belarus

It is well defined that high iodide doses (HID) induceapoptosis of thyroid cells. Thyrocyte programmed celldeath is possibly due to oxidative stress. This study wasaimed at investigation of HID influence on thyroidfunctional activity and its pro/antioxidant status.

All experiments were performed on female Wistarrats, weighting 180–200 g (n=10). Rats were treatedwith potassium iodide 0.7, 7 and 70 mg/kg weight(10, 100 and 1000 physiological doses, respectively)and were sacrificed after 24 hours. As a control weused animals receiving daily physiological iodinedose (0.07 mg/kg). Thyroid cell functional activitywas determined by measuring general, free and pro-tein-binding tissue iodide levels and thyroperoxidaseactivity. Pro/antioxidant status was estimated bymeasuring the levels of thiobarbituric acid reactivesubstances (TBARS) and the activities of catalase,

superoxide dismutase (SOD) and glutathione reductase(GR).

Our data indicate that HID inhibit thyroid functionalactivity on two levels. Firstly, 1.58- and 1.18-fold reduc-tion of total iodine level (7 and 70 mg/kg KI, respec-tively) and 1.30-fold reduction of free iodide levels (7mg/kg KI) evidences a suppression of iodide uptake.Secondly, this effect was accompanied by inhibition ofiodine inclusion into organic compounds: HID de-creased thyroperoxidase activity 3.98-fold (70 mg/kgKI) and reduced the protein-bound iodine levels 1.12-,2.19- and 1.23-fold (0.7, 7 and 70 mg/kg KI, respec-tively). Interestingly, KI treatment (70 mg/kg) lead to asignificant (1.18-fold) increase of thyroid weight. HIDtreatment caused an increase of tissue TBARS levels(1.14, 1.14 and 1.25-fold) and augmentation of catalaseactivity (1.17, 1.26 and 1.51-fold) in rats treated with


0.7, 7 and 70 mg/kg KI, respectively. GR activity wassignificantly (1.21-fold) increased only in rats treatedwith 70 mg/kg KI, and SOD activity was not altered.The significant increase in catalase activity may becaused by induction of H2O2 production in thyroid cellsby HID.

Our results suggest that HID have a toxic effect on thy-roid, since even single KI treatment inhibits thyroid cellfunctional activity by suppression of iodide uptake and ofiodine incorporation into organic compounds. This effectseems to be accompanied by oxidative stress evidenced byaccumulation of products of lipid peroxidation.

89 Poster

Antioxidant effects of hydroxyl derivatives T-stilbene against the free radical inducedlipid peroxidation of LDL from human plasma

Henryka £apkowska, Wojciech Bogus³awski


The antioxidant effects of hydroxy-derivatives oftrans-stilbene, that is; 4-hydroxy-trans-stilbene (4-HS),diethylostilbestrol (DES), 3,4’,5-trihydroxy-trans-stil-bene (3,4’,5-THS, resveratrol) and 3,3’,4,5’-tetrahydr-oxy-trans-stilbene (3,3’,4,5’-THS, piceatannol), againstthe peroxidation of lipids of low-density lipoprotein(LDL) from human plasma was investigated.

The peroxidation was initiated by Cu2+ ions andstudied by monitoring the formation of malondial-dehyde (MDA). It was found that the antioxidant activ-ity of these compounds depends significantly on theposition of the hydroxyl groups.

90 Poster

Antioxidative effect of resveratrol on the prevention of experimental acute pancre-atitis

Micha³ £awiñski1, Zbigniew Œledziñski1, Micha³ WoŸniak2, Jolanta Juraniec-Kubasik3, Wojciech Bogus³awski2

1 — Katedra i Klinika Chirurgii Ogólnej i Transplantacyjnej, Akademia Medyczna w Gdañsku, ul. Dêbinki, 80-211 Gdañsk,2 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 3 — SamodzielnaPracownia Mikroskopii Elektronowej, Akademia Medyczna w Gdañsku, Gdañsk

The data published in 2002 by Gullo et al. point to therelation between etiology of acute pancreatitis and geo-graphic area. Cholelithiasis as an etiological factor pre-dominates in mediterranean countries whereas alcoholis a main etiological factor in the northern countries.Acute pancreatitis caused by alcohol is not a simpleconsequence of the amount alcohol consumed, becausein many countries leading in alcohol consumptioncholelithiasis is the predominant etiological factor.Analysis of data provided by WHO indicates that wineconsumption seems to be a more healthy custom thatthe drinking of high percent drinks. The reason for this“protective” effect of wine is unknown. It seems proba-ble that resveratrol, stilbene-derived compound, whichis present in wine, may protect the pancreas against theharmful properties of alcohol. The purpose of this studywas to examine the protective effect of resveratrol inexperimental acute pancreatitis (EAP). Material andmethods: EAP was induced in male Wistar rats by ret-rograde injection of 0.5 ml of 160 mM tert-butylhydroperoxide (ButOOH) solution into the bile-pan-

creatic duct (BPD). Male Wistar rats were randomly di-vided into 6 groups. In Groups 1 and 2 (control), 0.5 mlof physiological saline was injected into the commonbile pancreatic duct. In Groups 3 and 4 (rats with acutepancreatitis) 0.5 ml of 160 mM ButOOH dissolved inphysiological saline were injected retrogradely intoBPD over 15 minutes. In Groups 5 and 6, resveratrolwas administered intraperitoneally, 2 mg daily for 8days before pancreatitis induction. After having in-duced EAP observation period lasted 3 or 6 hours in theodd and even numbered groups respectively. After 3hours of observation the animals were killed by decapi-tation and examinations in light and electron micro-scope were performed. The results revealed profoundmorphological changes in the pancreas, namely: inter-stitial edema, acinar cell vacuolization, leukocyte infil-tration, focal necrosis of pancreatic acini and fat-tissuenecrosis. These changes were greater after 6 hours. Af-ter administration of resveratrol into peritoneal cavitythe changes in pancreata were less pronounced. Thesemorphological changes were confirmed by results of


biochemical investigations, namely: serum amylase ac-tivity, and the levels of carbonyl and sulphydryl groups.Natural resveratrol belong to the strong antioxidant

agents which may diminish oxidative stress and in con-sequence may have a positive effect in the therapy of ex-perimental acute pancreatitis.

91 Poster

Green tea protection against age-dependent ethanol-induced oxidative stress

Wojciech £uczaj, El¿bieta Skrzydlewska

Zak³ad Chemii Nieorganicznej i Analitycznej, Akademia Medyczna, ul. Mickiewicza 2, 15-230 Bia³ystok

It is known that ethanol intoxication leads to oxida-tive stress. In such a situation, potent antioxidants be-longing especially to natural products are investigated.The aim of this study was to investigate the influence ofgreen tea as a source of water-soluble antioxidants onthe antioxidative capacity of blood serum of aged ratschronically intoxicated with ethanol. 2, 12 and 24month-old male Wistar rats were used. The animalsfrom each age group were divided into the followingfour groups: 1. Control group (receiving control LieberDeCarli liquid diet for 5 weeks); 2. Green tea group (re-ceiving control liquid Lieber DeCarli diet containinggreen tea (7 g/l) for 5 weeks); 3. Ethanol group (receiv-ing control liquid Lieber DeCarli diet for one week andethanol Lieber DeCarli liquid diet for next 4 weeks); 4.Ethanol and green tea group (receiving control LieberDeCarli liquid diet containing green tea (7 g/l) for oneweek and then ethanol Lieber DeCarli liquid diet con-taining also green tea (7 g/l for 4 weeks). We found thatethanol intoxication caused an age-dependent decrease

in the activity of serum superoxide dismutase (up to70% of that of the 24-month control group), glutathioneperoxidase (to about 90%) and glutathione reductase(to about 85%) and a decrease in the levels of GSH (to75%), vitamins C and E (to 87% and 79%) and vitamin Aand �-carotene (to 70% and 57%, respectively). Changesin the serum antioxidative capacity were accompaniedby enhanced oxidative modification of serum lipid (in-crease in lipid hydroperoxides, malondiadehyde and4-hydroxynonenal level to about 170% of 24-month con-trol group) and protein (increase in carbonyl groupslevel to about 130% and 146%). Green tea partially pre-vented the changes in serum antioxidant parameterscaused by ethanol (most effectively in 12-months oldrats). Green tea significantly protected lipids and pro-teins against oxidative modifications (most effectivelyin 2- and 12-months old rats). The above results indi-cate that green tea effectively protects blood serumagainst oxidative stress caused by ethanol as well as byaging.

92 Poster

Effect of plant flavonoids on activity of cereal aphid catalase

Iwona £ukasik, Bogumi³ Leszczyñski

Katedra Biochemii, Akademia Podlaska, ul. Prusa 12, 08-110 Siedlce

Herbivorous insects evolved various resistance mech-anisms to plant prooxidants and oxygen free radicals.One of them is detoxication of toxic oxygen moleculesby antioxidant enzymes. These enzymes remove thefree oxygen radicals generated by plant prooxidantsand other endogenous reactions catalyzed by numer-ous oxidoreductases. The present paper reports on theactivity of catalase within tissues of two species of ce-real aphids: autoecious Sitobion avenae (F) andheteroecious Rhopalosiphum padi (L) and on effect ofcereal flavonoids quercetin and kaempferol on the ac-tivity of the enzyme.

Among studied morphs of the cereal aphids the high-est activity of catalase was recorded for winged adults

(alatae) and the lowest for larvae. Higher level of the en-zyme activity was found within R. padi tissues, that ishost-alternating between broad-leaved plants andgrasses, than in S. avenae confined only to grasses. Ac-tivity of the aphid catalase in vitro was significantly af-fected by the studied flavonoids. Both flavonoidsquercetin and kaempferol caused a decrease in the ac-tivity of the enzyme. The higher inhibition of thecatalase activity was found for quercetin. However, thelevel of inhibition was strictly associated with the ap-plied concentration of the flavonoids and exposuretime. The role of the plant flavonoids and the antioxi-dant enzymes in the chemical interactions between her-bivorous insects and their host plants is discussed.


93 Poster

An effort for intensification of antioxidant properties of fungal cultures

El¿bieta Malarczyk, Agnieszka Zmarz, Marcin Gr¹z

Zak³ad Biochemii, Uniwersytet M. Curie-Sk³odowskiej, Plac M.Curie-Sk³odowskiej, 20-031 Lublin

Natural substances with antioxidant properties arevaluable for possibility of food supplementation in theaim to counteract of the consequence of free radical dis-turbing in the organism. The fungi, especially medici-nal mushrooms are very interesting as the health sup-plementary products. Inter alias they are rich in lowmolecular substances active as antioxidants, amongthem phenolics and simple organic acids, but the mush-room material is more often tested for content ofanticancer polysaccharides than for anti free radicalscavengers possibilities. Our earlier studies show thepresence of some new substances with high anti freeradical capacity (Malarczyk et al., 2002). In the presentstudies 3 strains of medicinal fungi Inonotus obliquus,Ganoderma lucidum and Pleurotus cystidiosus weretested and the initial level of antioxidants was deter-mined. Subsequently the fungi were cultivated with theaddition of simple phenolics as guaiacol, vanillic andferulic acids as well as with the waste birch wood mate-rials after production of therapeutic betuline-rich prod-ucts from SYLVECO ([email protected]). We ex-pected that these supplements could act as stimulants

for increasing of antioxidant properties of fungal cul-tures. Antioxidant activity was measured in aqueousextracts of the cultures. Organic acids and phenolicswere determined by capillary electrophoresis.Production of peroxides was estimated withhomovanillate (PaŸdzioch-Czochra and Wideñska,1999). The results of the experiments allow to concludethat birch materials were the best stimulant, especiallywhen the fungi were cultivated under conditions ofsolid state fermentation. The best result was obtainedfor Ganoderma lucidum cultivated on the solid mediumrich in birch bark. Aqueous extracts of this funguscaused a near 100% inhibition of homovanillate oxida-tion. The MEKC method revealed new unidentifiedphenolic compounds in the post-culture materials of allfungal strains tested.References:Malarczyk E et al. (2002) Proc.XI Biennial Meeting of Society

for Free Radicals Research Intern, Paris, 430–433.PaŸdzioch-Czochra M, Wideñska A (1999) Analytica Chemica

Acta, 21: 177–184.

94 Poster

Effect of photodynamic therapy with hypericin in combination with light

Anna Malarska, Anna Marcinkowska, Jolanta Saczko, Agnieszka Chwi³kowska, Teresa Banaœ

Katedra i Zak³ad Biochemii Lekarskiej, Akademia Medyczna, ul. Cha³ubiñskiego, 50-368 Wroc³aw

Photodynamic therapy (PDT) is a process of selec-tive damage of malignant cells stimulated by thera-peutical agents, photosensitisers and light. Photo-sensitisers activated by visible light generatecytotoxic singlet oxygen and other reactive oxygenspecies (ROS). Several studies proposed PDT as an al-ternative way of treatment of multidrug-resistantcells.

Hypericin (a polycyclic aromatic naphthodianthrone)is a natural photosensitier produced in plants of the ge-nus Hypericum. This compound exhibits a potentphototoxicity both in vitro and in vivo. In the presentstudy we investigated the influence of hypericin in com-bination with laser light on the activities of antioxidantenzymes: superoxide dismutase (SOD) and catalase(CT) in cells in vitro.

The studies were performed on LoVo (doxoru-bicin-sensitive) and LoVoDx (doxorubicin-resistant) co-

lon adenocarcinoma cells. The cells have grown withvarius concentration of hypericin in the absence and inthe presence of light and were assayed immediately fol-lowing the irradiation or incubated for 30 min. Irradia-tion was carried out using red laser light (� = 632.8 nm)at a dose of 2.5 J cm–2. Cell viability was assessed 2 h af-ter irradiation using MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric as-say, which measures the capacity of mitochondrialdehydrogenases of viable cells to reduce MTT to a pur-ple formazan precipitate.

SOD activity measured 30 min after irradiation wassignificantly (by 40–45%) increased as compared to con-trol. Similar to the laser-treated samples, cells exposedto hypericin in the dark demonstrated an increase inSOD activity (25–28%). CT activity was decreased inboth cell lines: by 15% in LoVo Dx cells and by 30% inLoVo 28% cells. No cytotoxicity was observed for a low


concentration of hypericine (10–8 M). A higher concen-tration of hypericine (10–5 M) was highly toxic to bothcell lines in the dark.

These results suggest an influence of hypericin incombination with irradiation on the generation of ROSand antioxidant enzymes response in both cell lines.

95 Poster

Antioxidative system in roots of pea plants treated with lead ions

Arleta Ma³ecka, Aneta Piechalak, Barbara Tomaszewska

Zak³ad Biochemii, Uniwersytet im. Adama Mickiewicza, ul. Fredry 10, 61-701 Poznañ

The destructive effect of heavy metal including Pb isvisible, among other things, in generating reactive oxy-gen species (ROS), such as: superoxide radicals,hydroxyl radicals and hydrogen peroxide. They damagemost cell components including proteins, membranelipids and nucleic acids. We have demonstrated the dy-namics of ROS production in hydroponically cultivatedroots of pea plants treated with 0.1 and 0.5 mM lead ni-trate in relation to the time of cultivation.

Low molecular compounds and enzymes detoxicatingRFT take part in the processes protecting cells againstagainst the effects of ROS. The dynamics of activitychanges of two basic antioxidative enzymes: super-oxide dismutase (SOD) and catalase (CAT), were deter-mined in roots of pea which were Pb-stressed and incontrol plants. The above enzymes showed the highest

activity in root cells of pea treated with 0.5 mM Pb. Atthe same time we studied the influence of lead on thecontent of acid-soluble thiols (glutathione andhomoglutathione) and the synthesis of phyto- andhomophytochelatins in roots of Pisum sativum. Thelevel of thiols was measured with the use of the HPLCtechnique connected with the postcolumn reaction withthe Ellman’s reagent. During the first several hours weobserved a decrease in the GSH level in cells by ca 20%followed by a recovery. Moreover we found a decreasein the activity of the key enzyme of glutathionebiosynthesis, -glutamylcysteine synthetase, in rootcells under the influence of Pb ions. We also observedchanges of the redox potential of root cells evidenced bya decrease in the value of the GSH/GSSG ratio duringprolonged exposure of pea plants to Pb ions.

96 Poster

Development of novel aromatic aminoxyl tools to detect reactive oxygen species inmetmyoglobin dependent oxidizing system

Wies³awa Mickiewicz1, Halina Zajworoniuk1, Tomasz Sarnowski1, Jerzy Nowak2, Lucedio Greci3,Micha³ WoŸniak1

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Katedra i Zak³adBiofizyki, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 3 — Dipartimento di Scienza dei Materiali e dellaTerra, Universita di Ancona, Ancona, Italy

We designed and synthesized aromatic nitroxides,namely [1,2-dihydro-2,2-diphenyl-4-ethoxy-quinone-1-oxide] (QAL), and [1,2-dihydro-2,2-diphenyl-4-carbo-xy-quinone-1-oxide] (C–QAL) in order to detect reactiveoxygen intermediates of peroxidative activity ofmetmyoglobin in the presence of hydroperoxides.

EPR measurements were carried out to confirm theconsumption of aromatic nitroxides during metmyo-globin catalyzed hydroperoxide (CumOOH) conversion

to corresponding radicals. A question whether chain-breaking antioxidants such as trolox can preventperoxyl radical mediated conversion of aromaticaminoxyl into diamagnetic N-oxides was investigated.Addition of hydroperoxides resulted in time and con-centration dependent decay of EPR signal of aminoxylwhile trolox was found to effectively prevent conver-sion of aminoxyl into N-oxides.


97 Poster

Serum homocysteine and MCP-1 are associated with abdominal aortic aneurysmsize

Barbara Millo1, Ireneusz Wiernicki2, Hanna Bukowska1, Barbara Górecka-Szyld3, Kornel Che³stowski1,Marek Naruszewicz1

1 — Chair and Department of Clinical Biochemistry and Laboratory Diagnostics, Pomeranian Medical University, ul. PowstañcówWlkp. 72, 70-111 Szczecin, 2 — Department of General and Vascular Surgery, Pomeranian Medical University, Szczecin,3 — Department of Diagnostic Radiology, Pomeranian Medical University, Szczecin

Objective: The risk of nonaneurysm cardiovascularmortality before and after surgery increased with ab-dominal aortic aneurysms (AAA) diameter. Surgery ofaneurysm with diameter smaller than 5 cm is not ad-vised. Chronic inflammation is observed in patientswith AAA but both clinical and biochemical assessmentremains insufficient. The purpose of this study was toexamine if elevated biochemical markers: homocys-teine (HCY), lipoprotein a [Lp(a)], monocyte chemo-tactic protein 1 (MCP-1), C-reactive protein (CRP), totalcholesterol (Ch), HDL-Ch, oxidized LDL (Oxy-LDL),triglyceride (Tg) can distinguish patients with higherrisk of cardiovascular diseases.

Methods: We examined 31 patients divided accordinganeurysm size: I gr. >5 cm (n=15) and II gr. <5 cm(n=16). Mean age was: 70.8 ± 6.8 in gr. I; 67.7 ± 8.6 in

gr. II (NS). AAA diameter as well as interluminalthrombus dimension (ITD) were assessed by an abdom-inal USG and MRI or CT. HCY levels were determinedby HPLC; MCP-1, Oxy-LDL by ELISA and CRP by highsensitivity ELISA technique.

Results: Mean AAA size was 6.08 ± 1.01 cm in gr. I,4.00 ± 0.54 cm in gr. II (p<0.01). Mean ITD was 1.16 ±0.60 cm in gr. I, 0.59 ± 0.32 cm in gr. II (p<0.01). MeanHCY was 18.42 ± 4.21 �mol/L in gr. I, 14.78 ± 3.36�omol/L in gr. II (p<0.01). HCY levels correlated signif-icantly with the ITD (r=0.42); MCP-1 with the ITD andwith AAA diameter (r= 0.57 and r= 0.45, respectively).

Conclusion: HCY and MCP-1 levels may be useful inmanagement of patients with AAA which are at highrisk of cardiovascular mortality.

98 Poster

Does selenium supplementation with organic selenosemicarbazides improveneutrophil phagocytic activity?

Irena Musik


Neutropenia in patients undergoing chemotherapy,as well as decreased interacellular bactericidal activityare both predisposing factors to hospital-acquired in-fection. Selenium deficiency in hospitalized patientswas demonstrated to impair the ability of neutrophilsto kill C. albicans and Gram-positive bacteria. The maintrends of selenium studies concern their most suitablechemical forms (inorganic vs. organic compounds), tox-icity limits and their effects on the immunologicalmechanisms.

The study was carried out on female SWISS mice di-vided randomly into the following 3 groups: Group A(Control) without Se supplementation, Group B receiv-ing sodium selenite dissolved in water and Group C re-ceiving organoselenic compound, 4-(o-tolyl-)-seleno-semicarbazide of p-chlorobenzoic acid. The compoundwas synthesized in our laboratory: 0.1 mol of o-tolyl

isoselenocyanate and 0.1 mol of p-chlorobenzoic acidhydrazide in 30 ml methanol were heated for 30 min.The sediment formed, after cooling, was crystallized 3times from 20 ml of 95% ethanol.

Neutrophil phagocytosis and NBT reduction were ex-amined after 10 days selenium supplementation at adose of 1 mg/kg body weigth daily.

After sodium selenite supplementation, phagocytosisreached 16.7 ± 1.6% in Group A, 29.0 ± 4.5% in Group Bvs. 30.8 ± 7.3% in Group C. NBT test showed 3.8 ± 1.5%,2.2 ± 0.9% and 0.9 ± 0.5% of positive cells in Groups A, Band C, respectively. Our results on stimulation ofphagocytic activity with two selenium compounds sug-gest that the organic selenosemicarbazide compound isbiologically more active and can significantly improveneutrophil function.


99 Poster

Oxidative stress inhibits iodine uptake and organification in thyroid cells

Liliya Nadolnik


Iodine deficiency leads to development of endemic andnodular goiter. However, recently an increase in the num-ber of noniodine-deficient pathologies of the thyroid wasnoted, which was a result of the influence of unfavourableecologic factors, including radiation effects. What is thethyroid pronounced sensitivity to the disturbance inecologic equilibrium due to? Our earlier studies show thatsingle and chronic radiation effects as well as emotionaland pain stress reduce the thyroid status, inhibitthyroperoxidase (TPO) and disturb iodine metabolism inthe rat thyroid. Moreover, the development of the nodularpathology is accompanied by activation of oxidative pro-cesses in the thyroid, which suggests participation offree-radical mechanisms. The main aim of our researchwas to study the effect of activation of oxidative process onthe iodine uptake and oxidation by thyrocytes of rat thy-roid organic culture in vitro. Fe2+/ascorbate at concentra-tions of 0.1x10–3 — 0.1x10–4 M was used as a prooxidantsystem. The iodine content in the medium was assessedwith cerium arsenite and TPO activity was measured spec-trophotometrically. The addition of prooxidants was ac-

companied by increased concentrations of stable aldehydelipid peroxidation products in the medium by2.88–6.76-fold. Under these conditions, the iodine uptakeby thyrocytes was almost completely inhibited within 2hours. A 31.1% decrease in TPO activity was also found in2 hours, at Fe2+ ascorbate concentration of 0.1x10–4 M. Athigher concentrations, TPO was inhibited by 30% after 5hours and by 61.55% after 8 hours. The TPO inhibitionand iodine uptake were reversible since after 24 hours theenzyme activity returned to the control values. The addi-tion of the 1x10–2 — 1x10–4 M H2O2 lead to 24-h inhibitionof iodine cellular uptake and a decrease of TPO activity by17.23–33.4%. The data obtained strongly suggest pro-nounced sensitivity of thyroid hormone synthesis in thethyroid to oxidative stress. The disturbed iodine uptake,as well as its oxidation and organification by thyrocytesseem to be the most important mechanism of thyroid func-tion impairment under the action of an unfavourableecologic factor.The research was supported by a grant from the Belarusian

Foundation for Fundamental Studies (Grant B01-343).

100 Poster

Anthocyanins in cardiology

Jan Niedworok1, Edward Kowalczyk2, Maria Kopff3, Anna Kopff4

1 — Zak³ad Farmakologii, Wydzia³ Lekarski, Uniwersytet Medyczny w £odzi, £ódŸ, 2 — Zak³ad Biofizyki i Fizjologii Cz³owiekaWydzia³ Wojskowo-Lekarski, Uniwersytet Medyczny w £odzi, Pl. Hallera 1, 90-647 £ódŸ, 3 — Zak³ad Chemii i BiochemiiKlinicznej Wydzia³ Fizjoterapii, Uniwersytet Medyczny w £odzi, Pl. Hallera 1, 90-647 £ódŸ, 4 — Klinika Kardiologii IMW,Uniwersytet Medyczny w £odzi, £ódŸ

Anthocyanins are among the most important wa-ter-soluble plant pigments. They belong to flavonoids andare derivatives of 2-phenylo-benzo-gamma pyrane. Theyhave antioxidant and anti-inflammatory properties and,therefore, may be potentially used in the treatment of oxi-dative stress, frequently augmented in cardiovascular dis-eases. It was also reported that the progression of athero-sclerosis was slowed down by polyphenol-reach diet. Oneof the richest sources of anthocyanins (560 mg/100 g offruits) are chokeberry (Aronia melanocarpa) fruits.

The aim of the study was to assess the effects ofanthocyanins derived from chokeberry fruits on some pa-rameters of redox balance in an animal model.

The study group consisted of twenty male Wistar rats;10 received pure water for 3 months, and the other 10 ofanthocyanins from Aronia melanocarpa (100 mg/l). After-wards, blood samples were collected for the assessment of

the (1) content of substances reacting with thiobarbituricacid (TBARS; acc. to Rice-Evans), (2) antioxidant status(Randox), (3) glutathione peroxidase activity (GSHPx)(Randox), (4) concentration of sulphydryl (SH) groups(Ellman method) and (5) nitrite concentration (using theGriess reaction, acc. to Marlett et al.) in the plasma.

Anthocyanins reduced significantly the content ofTBARS (1.26 ± 0.1 vs. 3.02 ± 0.4 �mol/l, p<0.001) andthiol protein groups (5.7 ± 0.61 vs. 7.9 ± 0.22 mmol/l,p 0.05), glutathione peroxidase activity (1213 ± 129 vs.1112 ± 166 U/l, p>0.05) and nitrite concentration (12.4 ±1.7 vs. 8.8 ± 0.3 �mol/l, p>0.05).

Conclusions. Anthocyanins from chokeberry decreaselipid peroxidation and therefore may be potentially usefulto combat oxidative stress.


101 Poster

NADPH oxidase participation in switching from apoptosis to necrosis

Edyta Niemczyk1, Jakub Kêdzior2, Micha³ WoŸniak2, Takashi Wakabayashi1

1 — Department of Cell Biology and Molecular Pathology, Medical University of Gdañsk, ul. Debinki 1, 80-211 Gdañsk,2 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk

Cells treated with menadione undergo apoptosis dueto oxidative stress. Menadione is a synthetic derivativeof vitamine K (VK3). It induces release of superoxideanion and hydrogen peroxide. This project is concen-trated on checking the source of reactive oxygen spe-cies (ROS). In order to examine this reactions humanosteosarcoma 143B cell line was treated with differentchemicals: antimycin A — an inhibitor of complex III ofmitochondrial respiratory chain, rotenone — an inhibi-tor of complex I, oligomycin — an inhibitor of mitochon-

drial ATP-ase and with NADPH oxidase inhibitors:difenyleneiodonium chloride (DPI), apocynin,N-vanillylnonanamide and staurosporine.

Cells were pretreated with above chemicals for 1 hand then exposed to 100 �M manadione. It is expectedthat inhibitors of respiratory chain complexes as wellas NADPH oxidase will allow to detect the source ofROS release especially the participation of NADPHoxidase in oxidative stress. Preliminary data supportsthis assumptions.

102 Poster

StPCS1, a phytochelatin synthase from Solanum tuberosum

Przemys³aw Nuc1, Katarzyna Nuc2, Tamara Chadzinikolau3, Andrzej Stroiñski3

1 — Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, ul. Miêdzychodzka 5, 60-371 Poznañ,2 — Department of Biochemistry and Biotechnology, Agricultural University of Poznan, ul. Wo³yñska 35, 60-637 Poznañ,3 — Department of Plant Physiology, August Cieszkowski University of Agriculture, ul. Wo³yñska 35, 60-637 Poznañ

The ability to synthesize phytochelatins (PCs), metalcomplexing, cysteine-rich peptides is an importantmechanism of heavy-metal detoxification and tolleran-ce during prolonged exposures of plants to heavy metalions (Stroiñski, APP 21, 175-188, 1999). Theirnon-translational synthesis from glutathione and re-lated thiols is elaborated by -glutamylcysteinedipeptidyl transpeptidase (phytochelatin synthase(PCS); EC 2.3.2.15). A few PCS genes have been iso-lated from plants and yeast species, and unexpectedly,from Caenorhabditis elegans.

In our initial effort to genetic characterisation of PCssynthase from Solanum tuberosum we have prepared acDNA library from cadmium treated plants and se-quenced several cDNAs, which potentially encode thefunctional enzyme(s). StPCS1 clone expressed in Esch-erichia coli appears to produce an active phytochelatinsynthase. Southern blot analysis revealed that Solanumtuberosum genome contains a family of several PCSgene or gene-like sequences.

103 Poster

Microsatellite instability and DNA repair in colon cancer

Tomasz Obtu³owicz

PAN, Instytut Biochemii i Biofizyki, 02-106 Warszawa

The main causes of colorectal carcinoma (CRC)inlude: (i) inflammatory processes and high fat diet re-sulting in an increase of DNA damage, among otherslipid peroxidation (LPO) derived etheno-DNA adductsformation; (ii) deficiency in mismatch repair (MMR) re-sulting in increased genomic instability.

We have compared microsatellite instability (MSI; in5 microsatellites: Bat26, Bat40, D2, D5 and D17), ex-pression of MMR proteins (MLH1, MSH2, MSH6) aswell as the activity of DNA glycosylases specific for1,N6-ethenoadenine (A) and 3,N4-ethenocytosine (C) intumour and normal colon epithelium obtained during


surgical intervention on 50 sporadic CRC patients inNorway.

Out of the 50 analysed CRC cases, in 6 patients dis-turbances of genomic stability and/or MMR defi-ciency was observed. Four of them revealed MSI in tu-mour tissue: two in all five microsatellites, one case(patient No. 4) microsatellite instability coincidedwith the dysfunction of the MLH1 protein. In one casewith MSI, MMR deficient has not been measured yet.In the other four cases there was no correlation be-tween microsatellite instability and expression ofMMR proteins.

DNA glycosylases were measured only in 2 out of 6patients who revealed either MSI or MMR distur-bances. For one of them (patient No.4), neither A- norC-glycosylase activity was detectable in normal and tu-mour colon epithelium. This patient had also defectiveMMR, thus the level of DNA damage could be signifi-cantly elevated in normal colon and in tumour than inother tissue measured in this study. Futher investiga-tion are now in progress to evaluate the role ofetheno-DNA adducts and DNA glycosylase activities inthe genomic instability in colon cancer.

104 Poster

Hypochlorite-mediated oxidation of tyrosine dipeptides

S³awomir Olszowski1, Przemys³aw P³onka2, Ewa Olszowska1, Dorota Kusior1, Aneta NiedŸwiedŸ1

1 — Instytut Biochemii Lekarskiej Collegium Medicum, Uniwersytet Jagielloñski, ul. Kopernika 7, 31-034 Kraków, 2 — InstytutBiologii Molekularnej, Uniwersytet Jagielloñski, ul. Gronostajowa, Kraków

Myeloperoxidase, a neutrophil enzyme, oxidizes thechloride anion to the hypochlorite (HOCl/OCl–), which,in turn, reacts with amino groups of peptides and pro-teins to form mono- and dichloramines. Chloramines,though less reactive than HOCl/OCl–, are able to chlori-nate/oxidize various compounds containing the phenolresidue. It has been shown that at the inflammationsite, proteins containing Tyr moiety form 3-chloro-tyrosine, 3,5-dichlorotyrosine, 3,4-dihydroxytyrosine(DOPA) and dityrosine (DT). At present, the knowledgeof the relative yield of these reactions is poor.

We examined a series of mono- and dichloramines ofdi- or tripeptides containing Tyr. The fluorescence spec-troscopy was used for the detection of DT (ex = 310, em= 410 nm) and DOPA (ex = 280, em = 320 nm) andUV/Vis differential spectroscopy for the assay ofchlorotyrosine derivatives. Additionally, the phenoxylradical formation was monitored by EPR X-band spec-troscopy. Our preliminary results revealed that theyield of products of Tyr chlorination/oxidation, as wellas the phenoxyl radical formation strongly depend on

the peptide sequence. Interestingly, the dichloraminesof all examined peptides seem more efficient in the pro-duction of DOPA whereas monochloramines in the in-duction of DT. It was also found that DT formation ispreferred in the case of N-acetyl-Tyr and tripeptide(Lys-Tyr-Lys). Moreover, the Tyr-Ala, but not Ala-Tyrchlorination resulted in DT formation. The yields ofchlorotyrosine derivatives were comparable for all ex-amined peptides. The DOPA production was elevatedin chlorinated N-acetyl-Tyr and Ala-Tyr. The strong sig-nal of phenoxyl radical was detected for chlorinatedN-acetyl-Tyr and Ala-Tyr dichloramines. SinceN-acetyl-Tyr chlorination leads to DOPA and DT forma-tion, it is likely that the ring chlorination/oxidation ismediated not only by mono- and dichloramines but alsowith unstable chloramides. It is also interesting thatonly in some sequences the chlorination of Tyr leads toelevated phenoxyl radical concentration. Thus, it couldbe expected that in proteins only specific Tyr residuescould mediate a phenoxyl radical formation and its in-teraction with other reactive oxygen species.

105 Poster

The influence of flavonoids and glutamine on antioxidant status of animals

Kazimierz Pasternak, Maria Szpetnar, Ma³gorzata Sztanke


Flavonoids are polyphenolic compounds, which com-monly occur in cultivated plants consumed by humans.

Catechin is found in black grapes, red wine and teawhile quercetin and naringenin are occured predomi-


nantly in citrus fruits. These compounds show antioxi-dant activity and they play a role mainly in scavengingfree radicals.

The purpose of our work was to determine the influ-ence of flavonoids and glutamine on the antioxidantstatus of animals intoxicated by toxic metals (lead andcadmium).

The experiment was conducted on Wistar male rats,weighting 170–200 g. The animals were divided into fif-teen groups, each of 5 rats.

Group I received redistilled drinking water and it wasa control group. Groups II–VIII obtained lead nitrate in(500 mg/dm3 of lead) and additionally Groups III andVI received 200 mg/dm3 of quercetin, Groups IV andVII 200 mg/dm3 of catechin and Groups V and VIII 200mg/dm3 of naringenin. Besides, groups VI, VII andVIII received additionally glutamine (30 mmol/dm3).Groups IX–XV were treated as Groups II–VIII but theyreceived instead of lead cadmium chloride (500mg/dm3 of cadmium) instead of lead.

The animals were on a normal diet (LSM dry food)and they got solutions ad libitum. After 6 weeks the ani-mals were anaesthetised with 0.5 ml of 5% ketamine.

Liver, kidneys and blood serum were excised, the tis-sues were homogenised in four volumes of 100 mMTris/HCl buffer pH 7.4. The homogenates were centri-fuged of 5000 x g for 20 minutes. Activities ofsuperoxide dismutase (SOD) and glutathioneperoxidase (GPx) and ascorbic acid concentration weredetermined in the supernatants. SOD and and GPx ac-tivities were determined using the RANSOD andRANSEL kits, respectively. Ascorbic acid concentra-tion was determined by a modified colorimetric methodof Kyaw.

Intoxication with lead and cadmium resulted in de-creases in activities of SOD and GPx and in the concen-tration of ascorbic acid.

All the flavonoids administered (quercetin, catechinand naringenin) caused a slight reduction of the esti-mated parameters in comparison with the controlgroup. Simultaneous application of glutamine with thetoxic metals and flavonoids did not increase the en-zyme activities and only slightly influenced ascorbicacid concentration both in blood serum and in the liverand kidney.

106 Poster

Selected antioxidative enzymes in tissues microsurgically removed from the vocalfolds in the course of treatment of patients with Reinke’s oedema

Katarzyna Paw³owska-Góral1, Tatiana Gierek2, Danuta Zbrowska-Bielska2, Piotr Wardas2

1 — Katedra Chemii Ogólnej i Analitycznej, Œl¹ska Akademia Med. Wydzia³ Farmacji, ul. Jagielloñska 4, 41-200 Sosnowiec,2 — Katedra i Klinika Laryngologii, Œl¹ska Akademia Medyczna, Katowice

It is known that reactive forms of oxygen (RFO) par-ticipate in the inactivation of proteinase inhibitors,which, via increase in the activities of respective en-zymes, can result in changes in the structure of theconnective tissue. Such a mechanism has been postu-lated for the damage of the connective tissue in thecourse of systemic diseases. Similar mechanisms can-not be excluded with respect to the formation ofchanges of the type of Reinke’s oedema of vocal folds.Reinke’s oedema is a pathology which is frequently ob-served in smokers and can be connected with the in-creased exposure of smokers to chemical substanceswhich generate RFO. The intensified formation ofRFO causes an increase in the activities of cellularantioxidative enzymes which are responsible for thepreservation of tissue oxidative homeostasis.

The aim of our research was to determine activities ofsuperoxide dismutase (SOD) and glutathione peroxidase(GPx) in the homogenates of tissues which were micro-surgically removed from the vocal folds in the course oftreatment of the patients suffering fromReinke’s oedema.

The material was obtained during microsurgicaltreatment of the patients suffering from Reinke’soedema in the Department and Clinic ofLaryngology of the Silesian University of Medicine.Activity of SOD was measured according toBeauchamp and Fridovich’s and that of GPx accord-ing to Paglia and Valentine. SOD activity was 0.46U/cm3 and GPx activity 188 U/dm3 (mean valuesfrom 9 samples).


107 Poster

The activity of antioxidative enzymes in the blood of rabbits exposed to the action oftetrachloromethane (CCl4)

Katarzyna Paw³owska-Góral1, Maria Wardas2, Ewa Adamek1

1 — Katedra Chemii Ogólnej i Analitycznej, Œl¹ska Akademia Med. Wydzia³ Farmacji, ul. Jagielloñska 4, 41-200 Sosnowiec,2 — Katedra i Zak³ad ¯ywnoœci i ¯ywienia, Œl¹ska Akademia Medyczna, ul. Jagielloñska 4, Sosnowiec

Tetrachloromethane is a commonly known hepato-toxic compound. Its toxic activity consists in generat-ing free radicals (trichloromethyl and trichloroperoxyl)in the cells. Cytochrome P-450 plays an important rolein these processes. Some reports on the hepatotoxicityof CCl4 demonstrated that CCl4 can exert a direct de-structive effect on cell membranes, causing increasedlipid peroxidation. It suggests that CCl4 biotransfor-mation processes can disrupt oxidative-reduction bal-ance of the body.

The aim of this study was to estimate the activities ofsuperoxide dismutase (SOD) and glutathiose

peroxidase (GPx) in the blood of rabbits exposed toCCl4.

Chinchilla rabbits were administered intragastricallya single dose of CCl4. The activity of alanine andaspartate aminotransferases in the serum of the ani-mals indicated acute hepatocellular damage. The activ-ity of SOD increased almost three times in comparisonwith the control group, whereas the activity of GPx de-creased by half in the blood of intoxicated rabbits.These changes in the activities of antioxidative en-zymes suggest that detoxication of CCl4 is accompaniedby generation of active oxygen species.

108 Poster

Analysis of antioxidant parameters in synovial fluids of patients with rheumatoidarthritis

A. Pietrzycka1, M. Stêpniewski1, M. Pukal2, B. Batko3, P. Zagrodzki4

1 — Radioligand Laboratory of the Pharmacy Faculty, Collegium Medicum, Jagiellonian University, Cracow, 2 — ClinicalDepartment, County Hospital, Brzesko, 3 — Clinical Department, J. Dietel’s Hospital, Cracow, 4 — Institute of Nuclear Physics,Department of Food Chemistry and Nutrition, Cracow

Rheumatoid arthritis is an autoimmune disease char-acterized by chronic joint inflammation with infiltra-tion of macrophages and activated T cells. Oxidativestress at the site of inflammation may contribute to thepathomechanisms of progressive joint destruction. An-tioxidant enzymes, superoxide dismutase (SOD) andcatalase (CT), non-enzymatic antioxidants (albumin,bilirubin and uric acid), and selenium and zinc play acrucial role in the defense against the effects of joint in-flammation.

The aim of the study was to measure total antioxi-dant capacity, activities of SOD and CT, and the levelsof non-enzymatic antioxidants in the synovial fluid,and to seek correlation between the antioxidant pa-rameters. Knee joint synovial fluid was obtained from90 patients with diagnosed rheumatoid arthritis (56women, 34 men, 19–78 years old) in order to deter-mine Ferric Reducting Ability of Plasma (FRAP), ac-tivities of SOD /Misra and Fridovich/ and CT /Aebi/,

and concentration of albumin, uric acid, zinc, iron andchloride anions.

The interrelations between the most relevant param-eters were investigated by means of principal compo-nents analysis (PCA). This statistical method requiresthat the parameters are normally distributed. For thisreason original parameters were transformed into log-arithms. In order to eliminate the influence of originalunits, all parameters were standardized for meanvalue equal zero and SD equal one.

Applying PCA to the group studied we achieved reduc-tion of dimensionality, i.e. the 7 original parameters werereplaced by 3 first principal components which repre-sented most of the total variance of the data set. The load-ings of the first component revealed the interactions be-tween CT, Fe, and Zn, whereas second component indi-cated that FRAP was greatly depended only on SOD. Ad-ditionally, we observed (from the third component), thatalbumin concentration correlated with the chloride level.


109 Poster

Molten globule-like state of cytochrome c displays high peroxidase activity with syn-thetic hydroperoxide

Patrycja Piórkowska1, Izabela Æwiklak1, Edyta Celmer-WoŸniak1, Wies³awa Mickiewicz1,Takashi Wakabayashi2, Jerzy Nowak3, Micha³ WoŸniak1

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Department of CellBiology and Molecular Pathology, Medical University of Gdañsk, ul. Dêbinki 1, 80-211 Gdañsk, 3 — Katedra i Zak³ad Biofizyki,Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk

Proteins can exist in living cell under physiologicalcondition in native as well as molten globule state(Bychkova et al. Chemtracts; Biochem Mol Biol, 1993).Cytochrome c in vitro can be transformed into moltenglobule state at low pH, denaturant guanidine chloride(GdmCl) or after exposition to high temperature andacidic phospholipids (Bychkova et al. Biochemistry,1996).

In the present study we investigated effect ofcardiolipin (main acidic phospholipid of inner mito-

chondrial membrane) as well as GdmCl on peroxidativeactivity of cytochrome c. We used (1,2-dihydro-diphe-nyl-4-ethoxyquinolino-N-oxide) QAL aromatic nitroxidein EPR time dependent spin decay method to inspectperoxidative activity of cytochrome c variants.Non-native molten globule state of cytochrome c wasfound to display an efficient conversion of ButOOHinto free radical species.

110 Poster

Radiotoxic effects of cancer cells irradiated in vitro on neighbour nonirradiated cells

Renata Polaniak1, Agnieszka Szurko2, Maria Wide³3, Zbigniew Maniakowski2, Ewa Birkner1,Waldemar Przybyszewski2

1 — Katedra i Zak³ad Biochemii Ogólnej w Zabrzu, Œl¹ska Akademia Medyczna w Katowicach, ul. Jordana 19, 41-808 Zabrze,2 — Zak³ad Fizyki Medycznej, Centrum Onkologii w Gliwicach, Wybrze¿e Armii Krajowej 15, 44-100 Gliwice, 3 — Zak³adRadiobiologii Doœwiadczalnej i Klinicznej, Centrum Onkologii Oddzia³ Gliwice, ul. Wybrze¿e Armii Krajowej 15, 44-101 Gliwice

Radiation damage can be induced in cells directly(macromolecular ionisation, mainly DNA) and indi-rectly (reaction of free oxyradical products of waterradiolysis), both ongoing in nanoseconds. Recently,the attention is paid to the radiotoxic action of irra-diated cells on their nonirradiated neighbours, socalled bystander effect. Mechanisms of this actionare much slower because it is caused by clastogenicand toxic agents released from irradiated cells. Ithas been shown that culture media in which cellswere irradiated induce chromosome damage, DNAmutation, micronuclei and apoptosis in non-irradiated cells. Molecular signals, probably proteinnature, emanated from irradiated cells induce sig-naling ways in nonirradiated cells leading to thementioned events.

In the present work, the bystander effect was ana-lysed on multicellular megacolonies of human mela-noma Me45 cells. Six megacolonies growing on one halfof culture flask were irradiated with 5 Gy of 60-Cogamma-rays at the dose rate of 0.773 Gy/min. The sec-ond half of the same flask with another sixmegacolonies was shielded by lead brickets, 6 cm thick.Immediately after irradiation culture medium was

changed. In a proper time, megacolonies were sepa-rately collected and analysed.

The estimated parameter were: malondialdehyde(MDA) concentration, enzymatic activities of superoxidedismutase (CuZnSOD) and glutathione peroxidase(GSH-Pox) and the numbers of micronucleated,apoptotic and necrotic cells.

Comparable MDA content (0.9–1.1 �mol/mg protein)was found in cells traversed by radiation andnontraversed (shielded) cells (control: (0.6–0.8�mol/mg protein). Similarly, activity of SOD increasedin both groups to about 10.8 ± 0.42 and 10.18 ± 0.17 ni-trate units/mg proteins in irradiated cells and shieldedneighbours, respectively, at 72 h. On the contrary,GSH-Pox activity was increased (26.0 ± 12.2 U/mg pro-tein) in radiation-traversed cells immediately after irra-diation and fluctuated during incubation time, being stillsignificantly higher than in cells non-traversed by radia-tion. Cytologic analysis showed a parallel increase of MNfrequency up to 48 h in both groups which was 4.5 ± 1.9%and 4.8 ± 0.9% in directly irradiated cells and shieldedneighbours, respectively, at 48 h. Whereas MN fre-quency further increased in irradiated cells, its declinewas observed in shielded cells at 72 h. Increase of


apoptosis frequency was also comparable in both groupsand reached its peak of 10.27 ± 0.59% and 11.57 ± 1.03%,respectively, at 48 h. Maximum increase of necrosis wasalso frequently seen at 48 h and was even higher innonirradiated cells (2.31 ± 0.38%) than in their irradi-

ated counterparts (1.32 ± 0.2%). the obtained results ver-ify the presence of the bystander effect. Its molecularmechanisms are still obscure and require further experi-mental explanation.

111 Poster

Plasma protein peroxidation and enzyme antioxidants in children with chronic hepa-titis (CHH)

Stefan Popadiuk1, Anna Liberek1, Joanna Renke1, Agnieszka Szlagatys1, Micha³ WoŸniak2

1 — Klinika Pediatrii, Gastroenterologii i Onkologii Dzieciêcej, Akademia Medyczna w Gdañsku, ul. Nowe Ogrody 1-6, 80-803Gdañsk, 2 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk

Reactive oxygen species (ROS) are involved in thepathogenesis of chronic inflammatory diseases. The in-tensification of free radical reactions is observed inchronic inflammation, especially if the antioxidant bar-rier is not efficient. The indispensable components ofantioxidant barrier are glutathione peroxidase (GPx)and Cu/Zn superoxide dismutase (SOD1). The effect ofboth ROS overproduction and parallel antioxidant en-zyme insufficiency results in oxidation of DNA, lipidsand proteins.

Aim of the study was to analyze the plasma proteinoxidation by the evaluation of carbonyls group contentand activation of GPX and SOD1 in erythrocytes of chil-dren with CHH. Material and methods: 47 children, 7to 18 years of age, with histopathologically confirmedCHH, were examined in order to assess 1) carbonyl con-tent in plasma proteins by the calorimetric methodwith 2,4-dinitrophenylhydrazine and 2) activities ofGPX and SOD1 in erythrocytes using the RANSEL and

RANSOD kits of Randox. The control group consistedof 61 healthy children aged from 6 to 18 years. Statisti-cal significance of differences was verified with theMann-Withney U-test. Carbonyl content [nmol/mg ofprotein] in the plasma of children with CHH (1.07 ±0.33) was higher than in the control group (0.86 ± 0.2,p<0.001). Activity of GPX [U/gHb] was lower in thegroup of children with CHH (18.7 ± 9.7) than in healthyones (26.1 ± 0.2, p = 0.005). A significant increase ofSOD1 [U/gHb] was found in children with CHH (2470 ±714) as compared to healthy controls (1857 ± 782,p=0.006). Conclusions: In patients with CHH intensifi-cation of free radical reactions can be observed, whichconfirms the role of ROS in its pathogenesis. One of thereasons for this phenomenon may be due to the inap-propriate activities of antioxidant enzymes. The induc-tion of SOD activity seems to be a response to increasedoxidative stress.

112 Poster

Protein oxidation products and plasma antioxidants in juvenile idiopathic arthritis

Joanna Renke1, Stefan Popadiuk1, Maria Korzon1, Beata Kaliñska-B³ach2, Ma³gorzata Szumera1,Micha³ WoŸniak2


The aim of the study was to find out if the protein oxi-dation products are cummulated in children with in-tense reactive oxygen species production due tochronic inflammatory process. Moreover, the changesin plasma antioxidants were studied together with thepossible correlations with the level of oxided proteins.A group of 14 children with diagnosed juvenile idio-pathic arthritis was studied. The level of plasma car-bonyl groups of protein, activities of superoxidedismutase (SOD) and glutathione peroxidase (GPx) and

total antioxidant status (TAS) were estimated in thegroup of children two times. The second examination(II) was made one year after the first one (I). The dis-ease activity was the same in both investigations. Car-bonyls were measured by the Levine method. SOD,GPx, TAS were estimated with the use of RANDOXkits. The carbonyl content in both investigations wasnearly the same — I: 1.69 ± 0.63; II: 1.64 ± 0.52nmol/mg of protein. The results obtained in antioxi-dant studies were as follows for Groups I and II, respec-


tively: SOD, 1982 ± 884 and 20611 ± 1483 U/gHb; GPx,19.4 ± 16.5 and 32.1 ± 23.1 U/gHb; TAS, 1.68 ± 0.22and 1.97 ± 0.34 mmol/l. Statistically significant in-crease in the level of TAS was found. An increase ofGPx activity was noted. Both results can be explainedby the successive treatment resulting in diminution ofthe inflammatory process and creating the possibility

to rebuild the antioxidant barrier functions. No correla-tions were found in this study between the plasma car-bonyls and antioxidants in children with JIA. Similarcarbonyl group content in the both investigations maybe due to the efficient mechanisms of proteolysis of oxi-dized proteins in children with JIA.

113 Poster

High concentrations of excised oxidative DNA lesions in human cerebrospinal fluid

Rafa³ Ró¿alski1, Piotr Winkler2, Daniel Gackowski1, Tomasz Paciorek1, Heliodor Kasprzak3, Ryszard Oliñski1

1 — Katedra i Zak³ad Biochemii Klinicznej, Akademia Medyczna, ul. Kar³owicza 24, 85-092 Bydgoszcz, 2 — Katedra i KlinikaNeurochirurgii, Akademia Medyczna, Bydgoszcz, 3 — Katedra i Klinika Neurochirurgii i Neurotraumatologii, AkademiaMedyczna, Bydgoszcz

The measurement of 8-oxoguanine (8-oxoGua) is re-garded as a good biomarker of oxidative DNA damage.Following excision from DNA the modified base(8-oxoGua) or nucleoside (8-oxo-2’-deoxyguanosine) isexcreted into urine or cerebrospinal fluid (CSF) wheretheir presence has been acknowledged to be reflectiveof an involvement of different DNA repair pathways.

In this study we have found that the excretion of8-oxo-2’-deoxyguanosine (8-oxodGuo) into CSF calcu-

lated per kg of brain weight per 24 h is 0.61 nmol whilethe excretion of the same compound into urine reachesthe value of 0.26 nmol/kg of body weight per 24 h. Ithas also been found that the ratio of 8-oxodGuo to8-oxoGua is about five times higher in CSF than in theurine. This suggests that in neuronal cells involvementof nucleotide excision repair pathway in removal of oxi-dative DNA may be more distinctive than that in an av-erage cell of the organism.

114 Poster

Pro- and antioxidative parameters in the kidney of rats with streptozotocin-induceddiabetes

Aleksandra Rudzik, Anna Stiepanow-Trzeciak, Anastasis Pacanis

Katedra i Zak³ad Analityki Klinicznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 7, 80-041 Gdañsk

Oxidative stress (OS) is defined as an increase in thesteady-state level of reactive oxygen species. It may oc-cur as a result of increased free radical generationand/or decreased antioxidant defence mechanisms.Oxidative stress is implicated in the pathogenesis ofmany diseases e.g. diabetes mellitus. Chronichyperglycemia is the primer of a series of cascade re-actions causing the overproduction of free radicals.There are growing evidences that oxidative stress maycontribute to the development of diabetic nephro-pathy. The aim of this study was to determine the lev-els/activities of oxidation products and antioxidantsin the kidney of rats with experimental diabetesmellitus.

Pro- and antioxidative parameters were measured inkidney homogenates of 2-month diabetic rats (Wistar).Diabetes was induced using streptozotocin (STZ; 65mg/kg, i.p.). Blood glucose was significantly higher in

the STZ group (488 mg/dl) relative to the control group(142 mg/dl). The following parameters were deter-mined: 1) prooxidative parameter: the level of a lipidperoxidation product (malondialdehyde, MDA) 2)antioxidative parameters: the levels of reducedglutathione (GSH) and oxidized glutathione (GSSG),and activities of glutathione peroxidase (GPx),superoxide dismutase (SOD), and glutathionetransferase (GST).

MDA content in the diabetic kidney was higher com-pared to the control group (0.12 nmol/mg protein vs.0.058 nmol/mg protein). Renal GSH concentration wasslightly decreased (0.94 nmol/mg protein vs. 0.50nmol/mg protein) but GSSG increased (0.06 nmol/mgprotein vs. 0,04 nmol/mg protein) in the diabetic rats.SOD and GPx activities in the diabetic kidneys were de-creased (401 U/mg protein and 156.6 mU/mg proteinvs. 531 U/mg protein and 277 mU/mg protein, respec-


tively). GST activity was similar to the control group(1.8 U/mg protein vs. 1.9 U/mg protein).

In conclusion diabetes promotes overproduction offree radicals in the kidney.

115 Poster

Microenvironmental factors modulating peroxynitrite action on porcine bloodplatelets

Tomasz Rusak, Halina Stelmach, Marian Tomasiak

Zak³ad Chemii Fizycznej, Akademia Medyczna, ul. Mickiewicza 2A, 15-230 Bia³ystok

Peroxynitrite (ONOO–) is a potent oxidant and nitrat-ing agent formed in the rapid reaction betweensuperoxide radicals and NO. The production of largequantities of ONOO– is expected to occur duringischemia-reperfusion injury, inflammation and neuro-degenerative diseases where the production of both NOand superoxide radicals is elevated. ONOO– was re-ported both to inhibit and to induce platelet aggrega-tion but the detailed mechanism of its action on thesecells has not been established. These studies were per-formed to determine which component(s) of the biologi-cal environment in which platelet are normally presentmodulate the action of ONOO–.

It was shown that ONOO– (50–250 �M) slightly di-minished platelet aggregation measured in wholeblood, was a potent inhibitor of aggregation inplatelet-rich plasma (PRP) and behaved as a weakstimulator of aggregation when the cells were sus-pended in Tyrode buffer. Peroxynitrite was also able toinhibit other platelet responses, such as adhesion, se-cretion and procoagulant response.

Addition of hemoglobin (5 �M) and tyrosine (50 �M)to the suspension of platelets in plasma (PRP) and inTyrode buffer attenuated the action of ONOO– onplatelet aggregation.

In whole blood ONOO– (250 �M) reduced the amountof total –SH groups by 12 ± 3% (p<0.001), in the PRPby 18 ± 4% (p<0.001) and in the platelet suspensions inTyrode buffer by 30 ± 4% (p<0.001).

We have demonstrated, using LDH release test as ameasure of plasma membrane integrity, that ONOO–

(250 �M) significantly disrupted the plasma mem-branes of washed platelets, slightly affected the mem-branes of platelets in PRP and was without any effecton the cells in whole blood.

Addition of albumin, reduced glutathione or smallamounts of plasma to the suspension of platelets inTyrode buffer reduced the proaggregatory effect ofONOO– and diminished the degree of the plasma mem-brane disruption. In the cells suspended in Tyrodebuffer addition of ONOO– evoked a relatively a weakcalcium signal (measured with Fura 2), which was lesspronounced after addition of albumin.

These results are interpreted to mean that in wholeblood physiological concentrations of ONOO– do not af-fect platelets due to the protective action of hemoglo-bin, albumin and other plasma components. TheONOO– induced aggregation of platelets suspended inTyrode buffer seems to be an artifact produced by dis-ruption of the platelet plasma membrane.

116 Poster

Relationship between concentrations of protein carbonyl groups and chosen antioxi-dants in serum of patients with chronic ischemia of lower limbs

Magdalena Rutkowska, Krzysztof Strzy¿ewski, Maria Iskra, Maria Pioruñska-Stolzmann

Zak³ad Chemii Ogólnej, Katedra Biochemii Klinicznej, Akademia Medyczna, ul. Grunwaldzka 6, 60-780 Poznañ

Atherosclerosis is a multifactorial disorder with numer-ous risk factors, e.g. smoking, hyperlipidemia, hyper-uricemia, diabetes, adiposity, high blood pressure, stress,infections, and immunological reactions. It has been es-tablished that oxidation of macromolecules accompaniesatherosclerosis. Proteins are increasingly recognized as amajor biological targets of oxidative modifications. Radi-cal-mediated damage to protein may be initiated by elec-

tron leakage, metal-ion dependent reactions and autoxi-dation of sugars and lipids. Free-radical-induced damageto one or several susceptible amino acids leads to creationof aldehyde or/and keto residues, which have beendefinied as carbonyl (CO) products. End-products of lipidperoxidation, such as malondialdehyde (MDA), are alsoinactivating agents, possibly via Schiff-base products, i.e.short-lived species formed by the reaction of CO products


with amines. As an effect, binding of apolipoprotein B(apoB) to its receptor is perturbed. It may lead to deposi-tion of LDL in vascular walls.

The study was performed on a group of patients withchronic arterial occlussion (AO) of the lower limbs. Thecontent of serum CO groups as an effect of oxidativeprotein modification was studied and related to thelevel of chosen antioxidants. Serum CO groups weremeasured with dinitrophenylhydrazine (DNPH). Activ-ity of ceruloplasmin was determined with o-dianisidineas a substrate. Uric acid concentration was measuredby the reaction with uricase.

The mean value of CO group content in sera of the pa-tients was found to be 0.43 ± 0.36 mmol/mg of proteinand was significantly higher than in the control group(0.16 ± 0.11). The CO content was related to the degreeof ischemia of lower limbs. The average oxidase activityof ceruloplasmin in sera of the patients (137.9 ± 54.6U/l) was higher than in the control group (101.8 ± 28.3U/l). Concentration of uric acid was 54.3 ± 15.0 g/l.There were no statistically significant correlations be-tween these parameters in the group studied.

The results indicate an enhanced oxidative damage ofserum proteins in AO group as compared with the con-trol group.

117 Poster

Assay of concentrations of antioxidative vitamins in blood plasma of patients withlarynx cancer

Maciej Rutkowski1, Krzysztof Grzegorczyk2, Alicja Lipka-Kociszewska3, Maria Kopff4

1 — Department Chemistry and Clinical Biochemistry, Medical University in £odz, Pl. Hallera 1, £ódŸ, 2 — Clinics ofGastroenterology of 5-th Clinical Hospital of the MU in £odz, Medical University in £odz, Pl. Hallera 1, £ódŸ, 3 — Department ofLaryngology of the Pirogows District Specialized Hospital in £odz, Medical University in £oŸ, ul. Wólczañska 191, £ód¿, 4 — Zak³adChemii i Biochemii Klinicznej Wydzia³ Fizjoterapii, Uniwersytet Medyczny w £odzi, Pl. Hallera 1, 90-647 £ódŸ

Vitamins A, C, E and beta-carotene (provitamin A)play an important physiological role as antioxidants.They prevent oxidative stress that may cause cell dam-age and consequently cause occurrence and develop-ment of numerous diseases, including cancer. Controlof the concentrations of those compounds in blood maytherefore provide clinically significant data.

The aim of this study was to assay concentrations ofvitamins A, C, E and of beta-carotene in blood plasmataken from patients suffering from larynx cancer recog-nised both clinically and histopathologically.

The study was conducted on 133 subjects of bothsexes divided into four groups: I (control), of 33 healthysubjects; II, of 33 patients with chronic tonsillitis; III, of33 patients with precancerous state of larynx, and IV,of 34 patients with larynx cancer. None of the subjectssmoked or took vitamin preparations.

Blood was collected from cephalic vein on potassiumoxalate as an anticoagulant from fasting subjects.Blood plasma was analysed for vitamins A, C and E andbeta-carotene.

We found a statistically significant decrease of con-centrations of the examined vitamins for all groups ofpatients. The highest decreases were observed in pa-tients with larynx cancer. The especially low levels of vi-tamin A in this group may be explained by the affinityof this compound to epithelium correlated with the de-velopment of cancer.

The results show significant role of vitamin A defi-ciency for larynx cancer. It may be a premise for itssupplementation and indication for rational nutritionas a natural source of the vitamin.The study has been carried out within the grant BW/40/2002

of the Military Medical University, and continued withinthe UM grant 502-17-019.

118 Poster

Localization of photosensitizing dyes, Photofrin II and hypericin, in malignant andnormal cells

Jolanta Saczko1, Marta Mazurkiewicz2, Agnieszka Chwi³kowska1, Anna Marcinkowska1, Anna Malarska1,Teresa Banaœ1

1 — Katedra i Zak³ad Biochemii Lekarskiej, Akademia Medyczna, ul. Cha³ubiñskiego, 50-368 Wroc³aw, 2 — Instytut Zoologiczny,Uniwerytet Wroc³awski, Wroc³aw

Photodynamic therapy (PDT) is a cancer treatmentinvolving incorporation of photosensitizing molecules

into malignant tumors and their activation with visiblelight. In photodynamic therapy (PDT), visible light acti-


vates a photosensitizing drug accumulated in tumorcells and leads to generation of reactive oxygen species(ROS) causing cell death and tumor ablation.

In this work we investigated the intracellular localiza-tion of two photosensitizers: Photofrin II andhypericin. Fluorescence microscopy studies of two ma-lignant cell lines (A459, BM) and normal 3T3 Balb cellswere performed after different times of exposure toPhotofrin II and hypericin.

The investigation did not show differences in thelocalization of both photosensitizers. Their distri-

bution within the cells was dependent on the timeof incubation. After 1 hour the dyes localized closeto the inner side of the plasma membrane. After 2hours we observed a diffuse distribution of the pho-tosensitizing drugs in the cytoplasm. 3 hours of in-cubation caused accumulation of the photosen-sitizers around the nuclear envelope. After 4 and24 hours the localization of photosensitizers didnot undergo particular alterations. The localiza-tion of photosensitizers was similar in both malig-nant and normal cell lines.

119 Poster

The effects of different organoselenium compounds on blood platelet lipidperoxidation

Joanna Saluk-Juszczak1, Halina Wójtowicz2, Krystian Kloc2, Barbara Wachowicz1

1 — Katedra Biochemii Ogólnej, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ, 2 — Instytut Chemii Organicznej,Biochemii i Biotechnologii, Politechnika Wroc³awska, Wybrze¿e Wyspiañskiego 27, 50-370 Wroc³aw

The aim of our study was to evaluate the effects of differ-ent synthethic organoselenium compounds: 7-azabenzi-soselenazol-3(2H)-one; 2-phenyl-7-azabenzisoselenazol-3(2H)-one; 2-(pyridyl)-7-azabenzisoselenazol-3(2H)-one;2-(5-chloro-2-pyridyl)-7-azabenzisoselenazol-3(2H)-one;bis(2-aminophenyl)-diselenide and ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one)) on the blood platelets:their activation induced by thrombin, lipid peroxidation(enzymatical and non-enzymatical) and metabolism ofplatelet thiols. To estimate the biological effects of thetested organoselenium compounds we preincubated (20min, 37oC) the platelets (suspended in Ca2+/Mg2+ freebuffer (15 mM Tris/HCl, 140 mM NaCl, 10 mM glucose,pH 7.4) with these compounds at the concentrations of 1,10, 100 and 1000 �M and then stimulated with thrombinor Fe2+. In the platelets we measured the level ofthiobarbituric-acid reactive substances (TBARS), as amarker of lipid peroxidation, and the level of thiols. Ourresults demonstrated that the concentrations oforganoselenium compounds used did not cause lysis of

platelets (estimated by LDH release). Five from the sixorganoselenium compounds tested caused a slight in-crease of the TBARS level (up to about 20%). Onlybis(2-aminophenyl)-diselenide distinctly reduced bothenzymatical (thrombin stimulated) and non-enzymatical(Fe2+-induced) peroxidation of platelet lipids in adose-dependent manner. After preincubation with thiscompound (1000 �M) the level of TBARS inthrombin-stimulated platelets decreased from 3.8 ± 0.1 to1.9 ± 0.15 nmol/mg of platelet protein (3.4 ± 0.1, 3.0 ±0.11 and 2.1 ± 0.12 nmol TBARS/mg of platelet proteinfor preincubation with 1, 10 and 100 �M compound, re-spectively). The level of TBARS induced by Fe2+ treat-ment decreased from 4.2 ± 0.11 nmol/mg of platelet pro-tein in control platelets to about 3 nmol/mg of plateletprotein for all tested concentrations of bis(2-amino-phenyl)-diselenide. This compound significantly in-creased the level of reduced forms of glutathione (byabout 25%), cysteine (by about 250%) and cysteinylglycine(by about 200%) in the platelets.Supported by grant from the University of Lodz.

120 Poster

Changes in activities of selected antioxidant enzymes in human erythrocytes ex-posed to anatoxin-APaulina Siciñska, Eliza Ciesielska, Wirgiliusz Duda

Katedra Biofizyki Ska¿eñ Œrodowiska, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ

Toxic blooms of phytoplankton occur frequently dur-ing several last years. It is the result of antropogeniccontamination of water reservoirs. Cyanobacteria pro-duce substances which are toxic for many organisms.

One of the toxins is the anatoxin-A which is producedby at least there genera of cyanobacteria includingAnabaena, Aphanizomenon and Oscilatoria. Thisneurotoxin is a potent neuromuscular blocking agent.


Cyanobacterial toxins influence many kinds of cellslike neurons, lymphocytes and fibroblasts causingtheir morphological and functional changes and in-ducing oxidative stress. These data prompted us tostudy the effect of cyanotoxins on human erythrocytesin vivo due to red blood cells are exposed to toxinsfrom the moment of their absorption by intestinal villito the transfer to other tissues.

Washed human erythrocytes were incubated withAnatoxin-a at concentrations of 1, 10, 100 and 1000 nM

for 1, 3, 12 and 24 hours. The correlation between theactivity of selected antioxidant enzymes: catalase,superoxide dismutase, glutathione reductase andglutathione peroxidase and the concentration of thetoxin and the time exposure was observed. The de-scribed disturbances of antioxidant enzyme activity af-ter exposure on anatoxin in vitro, could be used to pre-dict changes of enzyme activities in vivo, duringlong-term consumption of water from reservoirs wherecyanobacteria toxin is produced.

121 Poster

Mechanism of disturbances of collagen biosynthesis in human dermal fibroblastssubmitted to oxidative stressPawe³ Sienkiewicz, Jerzy Pa³ka

Zak³ad Chemii i Analizy Leków, Akademia Medyczna w Bia³ymstoku, ul. Kiliñskiego 1, 15-230 Bia³ystok

Oxidative stress accompanying the effect of reactiveoxygen radicals is one of the factors, which decreaseprotein biosynthesis and stimulate the process of its ca-tabolism. Cells treated with t-butylhydroperoxide, thecompound which generates free oxygen radicals, arecommonly used as a model of oxidative stress. Conflu-ent human dermal fibroblasts were treated with 30 �Mt-butylhydroperoxide for 1 hour during one day, 3hours during three days and 5 hours during five days,in order to evaluate the influence of short-lasting (1 and3 days) and long-lasting (5 days) oxidative stress on col-lagen biosynthesis. The biosynthesis of this protein de-pends on insulin-like growth factor receptor (IGFR) ex-pression and on prolidase activity, that is regulated by�1-integrin receptor. The endpoint of signal transducedby these receptors is activation of MAP-kinases. There-fore, we studied prolidase activity (colorimetricmethod), expression of prolidase, �1-integrin receptor,IGFI-R receptor, MAP-kinases (Western Immunoblot)and DNA biosynthesis ([3H]-thymidine incorporationassay). Collagen biosynthesis was evaluated by mea-surement of 5[3H]-proline incorporation into proteins,susceptible to the action of bacterial collagenase.

It has been found that oxidative stress inhibits colla-gen biosynthesis in human dermal fibroblasts in amanner dependent on time, at which the studied cellswere exposed to the oxidant. Long-lasting oxidativestress contributed to a decrease in collagen biosynthe-sis to 30% of the control value. Prolidase activity andthe expression of the enzyme decreased only in thesecells, which were treated with oxidant for a short time.In these cells prolidase activity decreased by about20%. As a result of long-lasting effect of t-butylhydro-peroxide, no further decrease in prolidase activity oc-curred; however, expression of the enzyme signifi-cantly increased, as demonstrated by Westernimmunoblot analysis. It has been found that thesephenomena are not related to the expression of�1-integrin receptor. However, long-lasting expositionof the cells to the effect of the oxidant drastically de-creased expression of IGFI-R receptor. This phenome-non is probably the main reason of a drastic decreasein collagen (up to 30% of the control level) and DNAbiosynthesis (up to 50% of the control level) infibroblasts submitted to oxidative stress.

122 Poster

Interaction of potassium channel effectors with skeletal muscle mitochondria

Jolanta Skalska, Grazyna Debska, Adam Szewczyk

Instytut Biologii Doœwiadczalnej, Polska Akademia Nauk, ul. Pasteura 3, 02-093 Warszawa

Potassium channel effectors can be divided into twogroups: inhibitors (sulfonylureas, 5-hydroxydecanoicacid) and activators (nicorandil, pinacidil, diazoxide). Apotassium channel, inhibited by ATP (K-ATP channel),was found in the plasma membrane of pancreatic

B-cells. Its inhibition causes plasma membrane depo-larization, which leads to insulin secretion from B-cells.Glibenclamide (one of the sulfonylureas), a blocker ofthe K-ATP channel, is used in the treatment of diabetesmellitus type II. A K-ATP channel with similar proper-


ties was characterized in mitochondria (mitoK-ATPchannel) of many cells (hepatic, skeletal muscle andneuronal cells). The aim of our experiments was tostudy the influence of K-ATP channel effectors on ionhomeostasis in mitochondria from rat skeletal muscle,in C2C12 cell line and in myotubes. It was observed,that glibenclamide (potassium channel inhibitor) in-duced a mitochondrial swelling (EC50=8.15 ± 2.5 �M),decreased the inner mitochondrial membrane potentialand evoked eflux Ca2+ from the mitochondrial matrix.These effects were blocked by an inhibitor of mitochon-drial megachannel, cyclosporine A. Moreover,glibenclamide accelerated respiration rate in the pres-

ence of substrates of complex I of the respiratory chain.It seems that glibenclamide activates megachannel inskeletal muscle mitochondria by increasing the sensi-tivity of the megachannel to Ca2+ ions. Recently, it wasfound that diazoxide (K-ATP channel opener/activator,which is used as an anti-hypertensive drug) protectsheart muscle cells against ischemic injury. The mecha-nism of diazoxide action is still unknown. We also ob-served the protective effect of diazoxide on C2C12 cellsand on myotubes, which were subjected to oxidativestress. This result was not observed in the presence of5-hydroxydecanoic acid, a K-ATP channel blocker.Work supported by the Nencki Institute of Experimental

Biology.

123 Poster

MnSOD and transcription factors AP-1, NF-�B in human colorectal cancer

Micha³ Skrzycki, Hanna Czeczot, Ma³gorzata Podsiad, Zofia Porembska


Manganese superoxide dismutase (MnSOD) is an im-portant element of the antioxidant barrier, protectingmitochondrial genome against damage caused by reac-tive oxygen species, especially superoxide radicals. Ap-propriate regulation of MnSOD expression is very im-portant for oxidative cell balance. Expression ofMnSOD changes in the presence of various oxida-tive-stress generating agents. In regulation of MnSODexpression participate transcription factors AP-1 andNF-�B. They are sensitive to reactive oxygen species,therefore it is possible that these transcription factorsare involved in MnSOD regulation in state of oxidativestress.

In the present work the levels of MnSOD protein,AP-1 and NF-�B transcription factors were determinedand compared.

Studies were conducted on specimens of primarycolorectal cancer, colorectal cancer liver metastasesand corresponding normal adjacent tissues obtained

from the same patients by surgery. Levels of MnSOD,AP-1, NF-�B were determined using Western blottingtechnique. Amount of proteins was measureddensitometrically. Antibodies anti AP-1 and anti NF-�Bwere purchased from Sigma, and antibodies antiMnSOD from Cappel. Activity of MnSOD was deter-mined according to Beauchamp and Fridovich [1].

The results indicate that activity of MnSOD is corre-lated with the level of MnSOD protein. The studiesshow distinct correlation between the levels of MnSOD,AP-1 and NF-�B in primary colorectal cancer andcolorectal cancer liver metastases. They demonstratethat MnSOD is regulated by the transcription factorsstudied, both in colorectal cancer and colorectal cancerliver metastases. Therefore transcription factors AP-1and NF-�B participate in the development of primarytumours and in liver metastases.References:1. Beauchamp C, Fridovich I (1971) 44: 276–287.

124 Poster

Disturbance/impairment of redox balance of the glutathione system in rat forebrainunder endotoxaemia

Vyacheslav Slyshenkov, Anna Shevalye, Andrei Moiseenok

Institute of Biochemistry, National Academy of Sciences of Belarus, BLK 50, 230009 Grodno, Belarus

It is well known that endotoxaemia is accompanied bythe development of inflammatory response in animalcells and tissue. Its characteristic features include also

suppression of intracellular protective mechanismsand of enzymes neutralizing cytotoxic products causedby penetration of bacteria or their degradation prod-


ucts, the exogenous pyrogens. However, the functionalstate of enzymatic and non-enzymatic protective sys-tems of nervous cells under endotoxaemia remains un-clear.

Wistar rats were administered with E. coli lipopoly-sacharide by subcutaneous injection of 100 mg/kg bodyweight and killed and examined 24 h later. Principal pa-rameters of the glutathione system and of the antioxi-dant capacity in rat forebrain homogenates andsynaptosomes were measured. It was found that low butsignificant increase in diene conjugates along with nochanges of the antioxidant capacity in homogenates andsynaptosomes of rat forebrain were marked under theconditions investigated. At the same time a significantincrease in glutathione reductase and transferase activi-ties in the homogenate (from 18.4 ± 0.9 to 37.2 ± 1.2

nmol NADPH/min/mg protein and from 43.2 ± 3.2 to59.1 ± 2.1 nmol DNPSG/min/mg protein, respectively)and in the synaptosomal fraction (from 33.3 ± 1.5 to 55.3± 1.9 nmol NADPH/min/mg protein and from 11.2 ± 0.1to 15.1 ± 0.8 nmol DNPSG/min/mg protein, respec-tively) were observed. The content of oxidizedglutathione increased in the homogenate and insynaptosomes (from 0.13 ± 0.02 to 0.28 ± 0.03 nmol/mgprotein and from 0.26 ± 0.02 to 0.34 ± 0.02 nmol/mg pro-tein, respectively), whereas that of its reduced form wasunchanged in both the homogenate and synaptosomes.

These results indicate that the initiation of endo-toxaemia in animals is accompanied by significantchanges of the activities of enzymes important for thefunctioning of the glutathione system and for maintain-ing of its redox balance in the neurons.

125 Poster

Bioactivation of nitroglycerin to nitric oxide (NO) and S-nitrosothiols in rat liver andevaluation of the coexisting hypotensive effect

Maria Soko³owska1, Marek Bednarski2, Barbara Filipek2, Lidia W³odek3

1 — Collegium Medicum, Uniwersytet Jagielloñski, ul. Kopernika 7, 31-034 Kraków, 2 — Instytut Farmacji, UniwersytetJagielloñski, ul. Medyczna 9, Kraków, 3 — Instytut Biochemii Lekarskiej, Collegium Medicum Uniwersytet Jagielloñski,ul. Kopernika 7, Kraków

The aim of the present studies was to investigate ni-troglycerin (NTG) bioactivation pathway in the liverafter various periods of its administration. We alsoattempted to elucidate the relationship between NOand SNT levels, and concentration of non-proteinthiols (NPSH) and intensity of peroxidative pro-cesses. Intravenous injections of nitroglycerin causean increase in nitric oxide and S-nitrosothiols (SNT)levels in the rat liver. The same intravenous NTG in-jections in the rats pretreated with 5-day intra-peritoneal NTG administrations lead to a drop in thelevels of the biologically active NO, SNT and NPSH,which is accompanied with inhibition of lipidperoxidation. It indicates development of toleranceafter such period of nitroglycerin treatment, con-nected with antioxidant action of NTG, which occursat the cost of pharmacological activity of the drug,i.e. with contribution of the released NO and SNT. Onthe other hand, long term NTG administration (for

10 and 17 days) induces adaptive processes, which al-low for maintaning of NTG, SNT and NPSH concen-tration in the liver at the control level, thus tolerancedoes not develop in spite of cosiderably enhancedlipid peroxidation. Effects of NTG bioactivation inthe liver, i.e. the levels of NO and SNT released fromit, after different periods of drug administration cor-respond with hypotensive effect, which is known tobe dependent on NTG biodegradation in the vascularendothelial cells. Summarizing, the changes in NOand SNT levels observed in the rat liver after differ-ent periods of NTG administration parallel alter-ations in the hypotensive effect. Depending on treat-ment, duration and dosage, nitroglycerin, as NO andSNT donor, can lead to transient tolerance, which inliver is accompanied by anyoxidant action. Howeverthe further NTG administration can enhanceperoxidative damage of the liver with unchanged lev-els of NO and SNT in this organ.

126 Poster

Effect of hydroperoxides on aromatase activity in choriocarcinoma cells

Ewa Soko³owska, Ryszard Milczarek, Anna Hallmann, Jerzy KlimekKatedra i Zak³ad Biochemii Farmaceutycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk

Introduction: Placenta is the main source of estrog-ens in pregnancy. Aromatase (CYP19) catalyzes the

rate-limiting step in estrogen biosynthesis. Estrogensin pregnancy regulate progesterone biosynthesis, pla-


cental metabolism of cortizol, blood volume and vascu-lar tone. Inhibition of estrogen biosynthesis can affectabove-mentioned processes. There is increasing evi-dence that in preeclampsia the level of lipidhydroperoxides and other kinds of reactive oxygen spe-cies in blood is elevated. The most probable source offree radicals in pregnancy is placenta. Since lipidhydroperoxides can damage some enzymes, we investi-gated the effect of synthetic hydroperoxides (cumenehydroperoxide and t-butyl hydroperoxide as analogs oflipid hydroperoxides) on aromatase activity inchoriocarcinoma cell culture. These cells have anaromatase activity and many similarities totrophoblast cells. Methods: JAR Cells (choriocar-cinoma) were from ATCC. Aromatase activity was mea-

sured by tritium release from 3H-1� androstendione.Oxidative stress was measured by flow cytometry withDCF-DA. Cells viability was calculated from LDH leak-age. Results: Cells were incubated 2 or 24 h withcumene or t-butyl hydroperoxide. After incubation withhydroperoxide aromatase activity was reduced in adose-dependent manner. In long- (24 h) but not inshort-time (2 h) viability was reduced, too. The drop inviability was lower than the drop in aromatase activity.Antioxidants partially reversed effect of hydro-peroxides on aromatase activity. Conclusions: On thebasis the results obtained, we propose that lipidhydroperoxides may contribute to aromatase activityinhibition in some pregnancy-associated diseases.

127 Poster

Changes in mitochondrial superoxide ion production in osteosarcoma 143B cells af-ter dihydroorotate dehydrogenase inhibitors treatment

Jan Spodnik1, Micha³ WoŸniak2, Mariusz Karbowski3, Narcyz Knap2, Takashi Wakabayashi4

1 — Katedra Anatomii i Neurobiologii, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Katedra i Zak³adChemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 3 — Department of Cell Biology andMolecular Pathology, Nagoya University School of Medicine, Nagoya, Japan, 4 — Department of Cell Biology and MolecularPathology, Medical University of Gdañsk, ul. Dêbinki 1, 80-211 Gdañsk

Leflunomide (LFM), an inhibitor of dihydroorotatedehydrogenase (DHODH), which plays a key role in thede novo synthesis of pyrimidine, affects also the shapeand number of mitochondria. The other inhibitors like5-fluoroorotate or lapachol do not express suchchanges.

Experiments were performed on human osteo-sarcoma cell line 143B cells and rat liver Rl-34 cell linekept in standard humidified atmosphere with 5% CO2at 37oC. Drugs were added in logarithmic growth phaseusing DMSO as a solvent. Measurements were per-formed using flow cytometry and confocal microscopybased on specific fluorescent dyes: Mitotracker GreenFM, CMTM Ros, 10-N-nonyl acridine orange, dihydro-ethidium and carboxy-DCF.

Treated 143B cells displayed elevated transient for-mation of superoxide ion quantified by flow cytometricanalysis of fluorescence of the oxidation product ofdihydroethidium, picked at 1 h. In confocal microscopyabout two fold increase in the amount of mitochondria

was observed. This finding was confirmed by flowcytometry, showing also dynamics of this process.Other inhibitors of the enzyme DHODH (lapachol and5-fluoroorotate) failed to induce the generation ofsuperoxide ion and the proliferation of mitochondria.In the presence of uridine, LFM-induced arrest in earlyS-phase of the cell cycle was corrected, but the genera-tion of superoxide ion and the proliferation of mito-chondria was not suppressed.

Disorganization of coordination of mitochondria andcytoplasmic signaling are often associated with abnor-mal accumulation of mitochondria in various models ofcell death. Those processes leading to biogenesis of mi-tochondria are complicated and largely unknown i.e. inherbimycin A treated cells an abnormal proliferation ofmitochondria has been reported

Collectively our results suggested important role ofoxidative stress in proliferative response of mitochon-drial systems.


128 Poster

An eight-week course of Mediterranean diet modifies the acitivity of superoxidedismutase and plasma levels of copper and zinc in renal graft recipients

Ewa Stachowska1, Maria Olszewska1, Iwona Noceñ1, Teresa Weso³owska2, Leszek Domañski3, BarbaraDo³êgowska1, Marek Myœlak3, Jacek Ró¿añski3, Kazimierz Ciechanowski3, Dariusz Chlubek1

1 — Katedra i Zak³ad Biochemii i Chemii, Pomorska Akademia Medyczna, ul. Powstañców Wlkp. 72, 70-111 Szczecin, 2 — Chairand Department of Clinical Biochemistry and Laboratory Diagnostics, Pomeranian Medical University, ul. Powstañców Wlkp.72, 70-111 Szczecin, 3 — Klinika Nefrologii, Transplantologii i Chorób Wewnêtrznych, Pomorska Akademia Medyczna,ul. Powstañców Wlkp. 72, 70-111 Szczecin

The progression of atherosclerosis in renal graft re-cipients is among the major factors reducing survivaltime. Changes in the rate and spectrum of free radicalreactions in these patients have been suggested. An im-portant factor controlling free-radical reactions in vivois the activity of superoxide dismutase.

Objective: The aim of this study was to determinewhether a Mediterranean diet modifies plasma levels ofcopper and zinc in recipients. In this respect it seemedinteresting whether the activity of erythrocytesuperoxide dismutase containing copper and zinc ionsis altered as well.

Material and Methods: The study group comprised20 renal recipients on a Mediterranean diet, while thecontrol group comprised 16 recipients on a normaldiet. SOD activity was measured spectrophotometri-cally. The concentration of zinc and copper was deter-mined by atomic absorption spectrometry.

Conclusions: SOD activities and Cu levels in plasmawere increased after eight weeks of Mediterranean diet(p<0.001). Apparently, the diet suppressed the produc-tion of free radicals and in consequence contributed tothe sparing of the enzyme and its cofactor.

129 Poster

SIN-mediated linoleic acid nitration in the presence of synthetic neuromelanins

Krystyna Stêpieñ, Alicja Zajdel

Department of Molecular Biology, Biochemistry and Biopharmacy, Medical University of Silesia, ul. Narcyzów 1,41-200 Sosnowiec

Under conditions of oxidative stress, such as inflam-mation or sepsis, the increased production ofsuperoxide anion and nitric oxide can lead to the forma-tion of peroxynitrite and other reactive nitrogen spe-cies, that are capable of modifying of a variety ofbiomolecules through oxidation, nitration andnitrosation reactions. There is some evidence that oxi-dative and nitrative processes are involved in patho-genic mechanism associated with neurodegenerativedisorders including Parkinson’s disease.

We have investigated the reactions of linoleic acidwith a peroxynitrite donor 3-morpholinosydnonimine(SIN-1) and the effect of model neuromelanins on theSIN-mediated reactions using reverse phase HPLC cou-pled with tandem mass spectrometry. MS analyses

were performed in negative ion ionization mode withmultiple reaction monitoring (MRM mode) of a se-lected ion pair (parent and daughter ions) characteris-tic for each compound analyzed. Treatment of linoleicacid with SIN-1 yielded both nitration and oxidationproducts that can be separately analyzed by HPLC at314 nm and 234 nm, respectively. Nitro-hydroxy-octadecadienoic acid (NO2HODEs) and nitro-oxo-octa-decadienoic acid isomers (NO2ODEs) were identifiedas the main nitrated derivatives of linoleic acid formedin the SIN-mediated reactions at physiological pH. Wedid not detect nitro-octadecadienoic acid among the an-alyzed nitration products, although we detected thiscompound when linoleic acid reacted with sodium ni-trite at acidic pH. SIN-mediated linoleic acid oxidation


*p< 0.001

led to the formation of isomeric hydroperoxides(13-HPODEs and 9-HPODEs) and hydroxy- andoxo-derivatives (HODEs, ODEs). Synthetic neuro-melanins derived from dopamine (DA-melanin) and5-S-cysteinyldopamine (CysDA-melanin) inhibited oxi-dation of linoleic acid by SIN in a dose dependent man-ner. The effect of the melanins on SIN-induced linoleicacid nitration depended strongly on the type of melanin

polymer, determined by the nature of its precursor. Ni-tration of linoleic acid was suppressed by CysDA-mela-nin (pheomelanin), but potentiated by DA-melanin(eumelanin). The stimulating effect of DA-melanin onNO2ODE and NO2HODE formation was not affected bythe presence of bicarbonate. These data suggest thatneuromelanin may modulate the nitrating and oxidiz-ing action of peroxynitrite.

130 Poster

Effect of thyroid state on lipid peroxidation and thiol groups concentrations in rab-bit tissues

Marta Stryjecka-Zimmer, Stanis³awa Szymonik-Lesiuk

Katedra i Zak³ad Biochemii i Biologii Molekularnej, Akademia Medyczna im. Prof. Feliksa Skubiszewskiego, ul. Lubartowska 85,20-123 Lublin

Recent research indicate that thyroid hormones arephysiologic modulators of both tissue oxidative stressand protein degradation and modulate oxidative dam-age to lipids, DNA and proteins.

The effects of altered thyroid states on lipidperoxidation and concentration of thiol groups in rab-bit tissues were examined.

Hypothyroidism was induced by administration of0.05% methimazole in drinking water for 21 days andhyperthyroidism was elicited by a 5 days treatment ofrabbit with T4 (200 g/kg body weight).

Malondialdehyde (MDA) levels and concentrations oftotal, protein-bound and nonprotein sulfhydryl groups

were measured in the homogenated liver, heart, kidneyand brain in euthyroid, hyperthyroid and hypothyroidrabbits.

The concentration of total and nonprotein sulfhydrylgroups was modified in the tissues of both rabbitgroups, most significantly in the liver and heart. Alter-ations in the MDA level in all examined tissues of therabbits were also found.

The results indicate that the susceptibility to oxida-tive stress was increased in all tissues of hypo- andhyperthyroid animals, but there are tissue variations inthe antioxidant capacity and effectiveness of cellulardefense systems.

131 Poster

Protein carbonyl group concentration and ceruloplasmin activity in serum of menwith chronic arterial occlusion of the lower limbs during the postoperative treatment

Krzysztof Strzy¿ewski1, Magdalena Rutkowska1, Maria Iskra1, Wac³aw Majewski2

1 — Zak³ad Chemii Ogólnej, Katedra Biochemii Klinicznej, Akademia Medyczna, ul. Grunwaldzka 6, 60-780 Poznañ, 2 — KlinikaChirurgii Ogólnej i Naczyñ, Akademia Medyczna, ul. D³uga 1/2, 61-848 Poznañ

Oxidative stress and reactive oxygen species (ROS)play an important role in the etiology and progressionof many human diseases. Carbonyl groups (CO) areproduced on protein side chains when they are oxi-dized, and carbonyl derivatives can be generatedthrough oxidative cleavage of proteins. Protein CO con-tent is actually the most general indicator and com-monly used marker of protein oxidation. Carbonylatedproteins has been detected in several human diseases.

The effects of inflammatory reaction, surgery, andthe progression of atherosclerosis on copper enzymeactivities have been widely discussed. Ceruloplasmin

(Cp), the main protein binding copper in plasma, is oneof the positive acute phase proteins (APhPs), whoseconcentration and activity in plasma increase followinginfection, inflammation or trauma.

The subjects of this study were 30 patients, males,aged 45–75 years, with chronic arterial occlusion ofthe lower limb (AO). All patients underwent arterio-graphy of the lower limbs and ultrasound measure-ment of ankle pressure, and were treated surgically.They were divided into two groups according to thedegree of ischaemia of the lower limbs: moderate orcritical.


The oxidase activity of Cp in serum was determinedaccording to the method of Schosinsky witho-dianisidine dihydrochloride as a substrate. Content ofprotein CO was determined using 2,4-dinitro-phenylhydrazine.

It was shown that the activity of Cp significantly in-creased after 1–4 days of postoperative treatment andreached the reference activity after 12 days innon-complicated cases. The content of CO groups in pa-tients before surgery (0.70 ± 0.47; range of 0.1–1.5

nmol/mg protein) was significantly higher in compari-son with the control group (0.16 ± 0.11 nmol/mg pro-tein). During the postoperative treatment changes ofthe CO content were negatively correlated with the val-ues found before surgery (–r>0.7; p<0.05).

The reverse relationship between the concentrationof CO and the activity of Cp was noticed indicating a re-covery of the balance between prooxidant and antioxi-dant factors.

132 Poster

Evaluation of the liver antioxidative system after administration of 5-formyl-tetra-hydrofolic acid

Elena Sudnikovich, Natalya Melnichenko, Tatyana Rudyak, Svetlana Zabrodskaya

Laboratory of Biochemistry of Membranes, Institute of Biochemistry, BLK-50, Grodno, Belarus

The present study was aimed at examination of theliver antioxidative system after administration of an ac-tive form of folate, 5-formyl-tetrahydrofolic acid(5-formyl-THF). The investigations were carried out onWistar male rats weighing 170–200 g. The animalswere divided into three groups receiving intramuscu-larly 5-formyl-THF at doses of 7 mg/kg (Group I), 17.5mg/kg (Group II) and 35 mg/kg (Group III) for 5 days.The control group was injected with 1 ml/kg of 0.85%NaCl solution. We found that glutathione reductase ac-tivity increased by 22 and 21% in Groups I and II, re-spectively (p<0.05). The GSH content of blood had afalling tendency in these groups.

Administration of 5-formyl-THF at a dose of 35mg/kg brought about an increase in the activity ofsuperoxide dismutase by 60% (p=0.021) and decreasedblood GSH concentration by 17% (p=0.045). The activ-ity of glutathione peroxidase (measured with t-butylhydroperoxide and cumene hydroperoxide as sub-strates) did not change in all the animal groups studied.It is proposed that after administration of high doses of5-formyl-THF, de novo GSH synthesis is disturbed as aresult of intensification of processes of homocysteineremethylation.

133 Poster

Impact of carbon centered free radical chemistry on proapoptotic function ofButOOH in rat thymocytes

Emilia Syta1, Ewa Kossowska2, Agata Zauszkiewicz1, Jakub Kêdzior1, Dorota ¯urawa3, Micha³ WoŸniak1

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Katedra i Zak³adBiochemii, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 3 — Katedra Biochemii, Uniwersytet Gdañski,ul. K³adki 24, 80-822 Gdañsk

There is currently much interest in the role of reactive ox-ygen species in oxidative stress and apoptosis. Howeverthe precise role of particular free radicals in these complexbiological phenomena are still ill defined. The aim of pres-ent study was to inspect antioxidant and antiapoptoticproperties of 4-OH-TEMPO in rat thymocytes under condi-tions of oxidative stress induced by ButOOH. ButOOH free

radical chemistry largely depends on cytochrome c depend-ent biotransformation into alkoxyl (�OCH3) radical fol-lowed by �-scission mediated formation of carbon centeredmethyl (�CH3) radical and finally peroxyl (�OOCH3) radicalspecies. 4-OH-TEMPO is able to scavenge only carbon cen-tered radical among this triad of radical species. Our dataclearly show that ButOOH induced peroxidation of


liposomal membrane (in cytochrome c consisting of sys-tem) and thymocyte cell membranes can be effectively pre-vented by 4-OH-TEMPO. As early as after 4 hours of incu-bation of thymocytes with ButOOH we observedupregulation of HtrA gene which encodes heat shock andoxidative stress induced serine protease HtrA1 localized incell membrane.

4-OH-TEMPO was also inspected with respect to itsability to inhibit apoptosis of thymocytes induced byButOOH and has been found to effectively inhibitthymocyte death.

Initial level of apoptosis which reach as much as 42%in the presence of ButOOH has been found to be dimin-ished to 25% after addition of 4-OH-TEMPO.

134 Poster

Determination of superoxide anion in mitochondria from barley roots

Bo¿ena Szal, Ma³gorzata Drozd, Anna Rychter

Zak³ad Bioenergetyki Roœlin, Instytut Biologii Eksperymentalnej Roœlin, Uniwersytet Warszawski, ul. Miecznikowa 1,02-096 Warszawa

Mitochondrial respiratory chain is the main source ofreactive oxygen species (ROS) in non-photosyntheticplant tissues. Superoxide anion radical is formed in mi-tochondrial electron-transport chain at the level ofComplex I and Complex III. Ubisemiquinone is formedduring the Q-cycle at both sites of inner mitochondrialmembrane resulting in O2

.– generation toward the mi-tochondrial matrix and the intermembrane space.

The relatively short half-time of ROS and the efficientsystems evolved to scavenge them require that ROS de-tection techniques must be sensitive enough to effec-tively compete with these intracellular antioxidantcompounds. Additionally, reagents used for analysis ofROS must have access to the sites of their generation.

Aeration after anaerobiosis (post-hypoxia) appears tobe favorable for the generation of ROS. Symptoms ofoxidative stress were observed in several plant tissuesduring post-hypoxic period. The purpose of this workwas to compare superoxide anion production by controlmitochondria and mitochondria isolated after a periodof hypoxic treatment.

Production of superoxide anion by mitochondria iso-lated from barley (Hordeum vulgare L) roots was mea-sured by SOD-inhibitable oxidation of epinephrine toadrenochrome. Measurements was performed in theabsence and presence of detergent (Triton X-100) todetermine both, matrix and intermembrane pools ofO2

.–.Assays in the presence of detergent doubled the rate of

epinephrine oxidation indicating that O2.– generated to-

ward mitochondrial matrix is not measurable when thedetergent is absent. Hypoxic treatment increased mito-chondrial activity but did not increase mitochondrialsuperoxide anion production. Inhibition of O2

.– produc-tion by exogenous GSH and stimulation by addition of1-chloro-2,4-dinitrobenzene (CDNB) which depleted en-dogenous mitochondrial GSH suggest that the lack ofaugmented mitochondrial superoxide production inhypoxia is the result of increase in antioxidants concen-trations.This work was supported by KBN grant 6P04C 04520.

135 Poster

Quinolinic aminoxyl as a molecular sensor of peroxidative activity of cytochromeP-450 2B1

Anna Szel¹gowska1, Jaros³aw Meyer-Szary1, Wies³awa Mickiewicz1, Halina Zajworoniuk1, Lucedio Greci2,Micha³ WoŸniak1

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Dipartimento diScienza dei Materiali e della Terra, Universita di Ancona, Ancona, Italy

Cytochrome P-450 2B1 induced by phenobarbital hasbeen found to display peroxidative activity with syn-thetic and natural hydroperoxidases. After inductionby phenobarbital treatment its microsomal concentra-tion reaches as much as 2 �M/mg protein in rat liverendoplasmic reticulum. We exposed isolated

endoplasmic reticular membranes to 20 mM ButOOHin the presence of 100 �M quinolinic aminoxyl (QAL).Conversion of ButOOH into radical species resulted infast decay of ESR signal of aminoxyl. Amphenone Bhighly specific inhibitor of cytochrome P-450 attenuateaminoxyl spin disappearing from the endoplasmic re-


ticulum membrane. This study shows that QAL may beused as highly specific and very sensitive molecular

sensor of free radical generation in the inside ofsubcellular membranes.

136 Poster

Plasma protein carbonyls content reflects clinical severity of asthma

Agnieszka Szlagatys1, Maria Korzon1, Stefan Popadiuk1, Joanna Renke1, Micha³ WoŸniak2


Background: Chronic airway inflammation is presentin asthma. So far, there is no peripheral blood markerwhich would correlate with clinical severity of asthma.Oxidative stress plays an important role in thepathogenesis of allergic inflammation. Protein car-bonyl content indicates the intensity of oxidativestress. The aim of the study was to investigate if airwayallergic inflammation results in plasma protein oxida-tion.

Material and methods: 91 children with stable mild(n=56) and moderate (n=35) asthma were included inthe study. Peripheral eosinophil blood count was nor-mal (1–5%) in 46 patients and above normal (>5%) in 45patients. Control group comprised of 30 healthy chil-dren. Plasma protein carbonyl content was evaluatedusing the Levine method. Results: Plasma protein car-bonyl content was significantly higher in asthmatic pa-

tients (1.05 nmol/mg protein, SD=0.26) than in healthycontrols (0.85, SD=0.21 nmol/mg of protein). Theplasma carbonyl content was found to be significantlyhigher in patients with moderate asthma than mildasthma (moderate asthma: 1.12, SD=0.21 vs. mildasthma: 1.00, SD=0.27 vs. control: 0.85, SD=0.21nmol/mg of protein). There were no differences in theplasma protein carbonyl content between the group ofpatients with above normal blood eosinophil count(1.00 nmol/mg of protein) as compared to the group ofpatients with normal peripherial blood count (1.11nmol/mg of protein). Conclusions: The intensity ofplasma protein modification may be helpful in the diag-nosis and management of asthma. It may be of specialuse in young children where pulmonary function testsare impossible to perform.

137 Poster

Changes in the levels of lipid peroxidation, thiol groups and ascorbic acid in rat tis-sues after carbon tetrachloride intoxication

Stanis³awa Szymonik-Lesiuk1, Marta Stryjecka-Zimmer1, Gra¿yna Czechowska2, Maria S³omka2, AgnieszkaM¹dro2, Marian Wielosz3

1 — Katedra i Zak³ad Biochemii i Biologii Molekularnej, Akademia Medyczna im. Prof. Feliksa Skubiszewskiego,ul. Lubartowska 85, 20-123 Lublin, 2 — Katedra i Klinika Gastroenterologii, Akademia Medyczna im. prof. FeliksaSkubiszewskiego w Lublinie, Lublin, 3 — Katedra Farmakologii, Akademia Medyczna im. prof. Feliksa Skubiszewskiegow Lublinie, Lublin

Background. The aim of this study was to determinepossible relationships between the levels of lipidperoxidation, thiol groups and ascorbic acid after ad-ministration of carbon tetrachloride (CCl4), withoutor with N-acetylcysteine, a precursor for glutathionesynthesis.

Methods. Male Wistar rats weighing 200–250 g wereused in the experiments. The animals were divided into4 groups: I (control) group, was injected with olive oil,Group II received CCl4, 0.5ml/kg i.p. (5 mmoles) in ol-ive oil for 1 day, Group III received N-acetylcysteine (10mg/kg) for 1 day, and Group IV received N-acetyl-cysteine and CCl4 in olive oil. Animals were decapitatedafter 24 hours. The content of malondialdehyde (MDA),

thiol groups and ascorbic acid was determined in thebrain, liver, kidneys, heart and lung homogenates.

Results. Basal levels of MDA in all the examined tissueswere 1.1 ± 0.2 to 1.4 ± 0.4 nmol/mg protein. Administra-tion of CCl4 caused a two-fold rise in the MDA level in theliver (2.7 ± 0.8 nmol/mg protein) and brain (2.1 ± 0.7nmol/mg protein), but no statistically significant changesin the heart (1.4 ± 0.4 nmol/mg protein), kidney (1.6 ± 0.6nmol/mg protein) and lungs (1.5 ± 0.5 nmol/mg protein.N-acetylcysteine decreased the MDA level after CCl4 in-jury in all examined tissues. No significant changes in thelevel of thiols were observed in the liver (3.1 ± 0.9 vs. 2.9 ±0.9 �mol/g tissue) and lungs (0.9 ± 0.3 vs. 0.9 ± 0.8�mol/g tissue) after CCl4 and CCl4 + N-acetylcysteine ad-


ministration, but significant elevations were found in thebrain (0.6 ± 0.1 vs. 0.3 ± 0.1 �mol/g tissue) and heart (2.2± 0.4 vs. 1.6 ± 0.6 �mol/g tissue). The CCl4 injectioncaused reduction of ascorbic acid level in the liver (3.9 ±1.0 vs. 8.2 ± 1.3 mg/g tissue) and heart (0.9 ± 0.4 vs. 1.4 ±0.5 mg/g tissue) and its increase in the brain (1.4 ± 0.4 vs.0.9 ± 0.3 mg/g tissue), kidney and lungs (3.8 ± 1.1 vs. 2.1 ±0.9 mg/g tissue).

Conclusions. These results suggest that increasedlipid peroxidation and alterations of the levels of thethiol groups and ascorbic acid may be the primarymechanism of the toxic effect caused by free radicalsformed during the CCl4 intoxication. Administration ofN-acetylcysteine and ascorbic acid may play a role inthe treatment of the toxicity of CCl4.

138 Poster

Model neuromelanins suppress interleukin-6 production in cultured endothelial cells

Irena Tam, Barbara Skop, Krystyna Stêpieñ

Department of Molecular Biology, Biochemistry and Biopharmacy, Medical University of Silesia, ul. Narcyzów 1, 41-200 Sosnowiec

Neuromelanin, the dark pigment present in the cyto-plasm of most nigrostriatal neurons of the human brain,modulates neurotoxic processes under oxidative stressconditions. Recently, it has been suggested that neuro-melanin can act as a mediator of chronic inflammation inthe substantia nigra by influence on proinflammatorycytokin production.

We examined the effect of model neuromelanins(DA-melanin and CysDA-melanin) on the production ofIL-6 in endothelial cell cultures after stimulation withIL-1.

The neuromelanins were produced by autoxidation ofdopamine (DA-melanin) or enzymatic oxidation of do-pamine and L-cysteine in the presence of tyrosinase(CysDA-melanin).

Human umbilical vein endothelial cells (HUVEC)were isolated and cultured using a modified standardprocedure.

Cell proliferation in the culture modified by differentneuromelanin concentration was tested by CyQuant®Cell Proliferation Assay Kit (Molecular Probes).

For inducing IL-6 synthesis, cells were cultured in en-dothelial cell medium with or without recombinant hu-man IL-1� (1 ng/ml). After 1 h of incubation with thestimulant, the neuromelanins were added to culture atconcentrations of 5, 10, and 30 �g/ml. The levels of IL-6

in the culture supernatants were measured by ELISA(Quantikine®, R&D Systems). For normalization, thevalues were calculated per DNA content in the culturesand compared with control.

The obtained results showed that the presence ofCysDA-melanin in the culture without stimulant (IL-1)did not influence the IL-6 production. Under the sameconditions DA-melanin increased IL-6 secretion (88 ± 1pg/ml in the absence of melanin and 751 ± 5 pg/ml atDA-melanin concentration of 30 �g/ml).

The melanins tested decreased production of IL-6 instimulated endothelial cells; however, the inhibition ef-fect was more evident in the CysDA-melanin modifiedculture. Both neuromelanins caused a dose-dependentreduction in the amount of secreted IL-6. Exposure toCysDA-melanin at concentrations of 5, 10 and 30�g/ml resulted in 13, 18 and 31% reduction of IL-6 se-cretion, respectively. The IL-6 levels in the culture

supernatants of cells treated with DA-melanin at 5 and10 �g/ml were lower than those in the control but 30�g/ml DA-melanin stimulated release of IL-6.

Production of IL-6 by HUVEC cultured with IL-1(pg/ml; n=12).

These results suggest that neuromelanin could partic-ipate in the regulation of inflammatory cytokin activ-ity.


139 Poster

Proapoptotic function of hsp90 in 2-methoxyestradiol-induced cell death

Anna Trzmiel


2-Metoxyestradiol (2ME), mammalian, endogenousmetabolite of 17�-estradiol is present in human bloodand urine and has been reported to inhibit endothelialcell proliferation. It has been also reported that 1 �Mconcentration of 2-ME induces apoptosis of osteo-sarcoma 143B cells.

Heat shock proteins (HSPs), highly conserved pro-teins family among all organisms, accumulate in cellsexposed to heat and variety of other stressful stimuli.HSPs which function mainly as molecular chaperones,allow cells to adapt to gradual changes in their environ-ment and to survive in otherwise lethal conditions. Theevents of cells stress and cell death are linked andHSPs induced in response to stress appear to functionat key regulatory points in the control of apoptosis. Pro-teins of the Hsp90 family are constitutively expressed

in mammalian cells and make up 1–2% of cytosolic pro-teins. Their expression can be further stimulated bystress. They associate with signaling proteins includingligand-dependent transcription factors such as steroidreceptor, tyrosine kinases v-Src and serine/threoninekinases, and promote conformational maturation ofthese proteins.

143B osteosarcoma cells were treated with 1 �M 2MEalone and with 1 �M geldanamycin during 24 h. Thenamount of DNA (apoptosis level) was examined afterpropidium iodide staining of the cells, using a flowcytometer. Cell cycle arrest in subG1 phase was ob-served. We demonstrated that geldanamycin, a phar-macological depletor of Hsp90, significantly attenuatedapoptosis induced by 2ME. The results obtained sug-gest that Hsp90 has a proapoptotic function.

140 Poster

Methimazolå does not exhibit antioxidant effect in thyroid gland tissue of rats ex-posed to single external gamma irradiation

Olga Valentsiukevich, Zoya Niatsetskaya, Liliya Nadolnik


It is well known, that the major action of methimazole(MMI) is to inhibit synthesis of thyroid hormones in thethyroid gland. However, recent studies have shownthat MMI has also antioxidant and immunomodulatoryeffects (Ho Kim et al., 2001).

In this study, the activity of lipid peroxidation pro-cesses and antioxidant defenses were measured in thethyroid gland of rats received MMI at doses of 2.5 and10 mg/kg body weight for a 2-week period. The influ-ence of single external gamma-irradiation at a dose of 1Gy on the animals receiving the same MMI doses wasalso studied. The MMI-induced hypothyroidism was ac-companied by a pronounced activation of oxidative pro-cesses in thyroid cells. This conclusion is based on in-creased activities of catalase (Cat, by 23.58%) andsuperoxide dismutase (SOD, by 29.4%) and by elevatedconcentration of thiobarbituric-acid reactive sub-stances (TBARs; by 37.5%) in rat thyroid. However, thiseffect was observed only in pronounced hypo-thyroidism (10 mg/kg MMI). The radiation exposurelead to a 1.34-fold increase of TBARs concentration in

the group of control animals. Single gamma-irradiationwith a dose of 1 Gy may have an inhibitory effect on theantioxidant system since we have not found any in-crease of Cat and SOD activities either in the thyroidtissue of control group rats, or in animals receivingMMI. Under such conditions, a significant increase ofTBARs level (1.69-fold) was observed at a MMI dose of10 mg/kg. These results show that the MMI-inducedhypothyroidism does not stimulate the function of theantioxidant system in the thyroid tissue of irradiatedrats. Moreover, histological examination of the thyroidgland of irradiated animals receiving MMI at a dose of10 mg/kg revealed an area with lymphoid auto-aggresion.

The obtained results indicate a complicated mecha-nism of the effect of MMI on the metabolism and anti-oxidant activities of thyroid cells. We suggest that theenhanced lipid peroxidation in MMI-inducedhypothyroidism results in destruction of thyrocytes, in-crease of the level of thyroid autoantigen in the bloodand development of autoimmune aggression.


141 Poster

Resveratrol may protect blood platelets against changes in thiols induced by plati-num compounds used in chemotherapy

Barbara Wachowicz1, Beata Olas1, Edward Bald2, Rafa³ G³owacki2

1 — Katedra Biochemii Ogólnej, Uniwersytet £ódzki, ul. Banacha 12/16, 90-237 £ódŸ, 2 — Zak³ad Chemii Œrodowiska,Uniwersytet £ódzki, £ódŸ

Cisplatin (cis-diamminedichloroplatinum II, cisPt) isespecially useful in the treatment of epithelial malig-nancies, however, the use of cisplatin is accompaniedby several toxicities including haematological toxicity.Contrary to cisplatin, selenium-cisplatin conjugate([NH3]2Pt(SeO3); Se-Pt) has only a slight toxic effect onblood platelet function. Thiols are involved in the mech-anism of platinum compounds action on platelets. Theaim of the present studies was to examine in vitro howtrans-resveratrol (trans-3,4’,5-trihydroxystilbene) actson the levels of platelet glutathione (GSH) and otherthiol-containing compounds and how, as an antioxi-dant, protects blood platelets against the oxidativestress caused by platinum compounds (cisPt andSe-Pt). To analyse the level of thiols in human bloodplatelets treated with platinum compounds in the pres-ence of resveratrol the classical HPLC technique hasbeen used. Blood platelets isolated by differentialcentrifugation of human blood were incubated (30 min,37°C) with cisPt or Se-Pt at dose of 10 mg/ml that in-hibits platelet function and with resveratrol (25mg/ml). The obtained results indicate that platinumcompounds caused in platelets a decrease of both re-duced glutathione (GSH) and free thiols: cysteine

(CSH) and cysteinylglycine (CGSH). The pool of thesecompounds in unreduced form was increased. Plati-num compounds also caused a diminution of plateletprotein thiol content. Resveratrol (after 30-min action)at the concentration of 25 mg/ml reduced partly the de-crease of platelet thiols, particularly thiols inacid-soluble fraction. The observed behaviour of thiolgroups in blood platelets showed that oxida-tion-reduction process may play an important role inthe biological function of these cells. Potential thera-peutic strategies should be taken into account the in thepreventing of changes of cellular thiols induced bysome anti-cancer drugs (including platinum com-pounds). In agreement with this hypothesis, the com-pounds present in human diet (resveratrol and sele-nium compounds) may have a protective action andthey may minimise the toxicity and side effects of thechemotherapeutic agent without effecting its anti-tumor activity. Here, we showed that in blood plateletsresveratrol has protective effects on changes in the lev-els of different thiols (glutathione, cysteine andcysteinylglycine and thiol groups in proteins) inducedby platinum compounds.

142 Poster

Regulation of vascular endothelial growth factor synthesis by doxycycline regulatedoverexpression of heme oxygenase-1

Joanna Wêgrzyn1, Agnieszka Siaœkiewicz1, Krystyna Staliñska1, Alicja Józkowicz2, Józef Dulak1

1 — Department of Cell Biochemistry, Faculty of Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków,2 — Department of Molecular Genetics and Genetic Engineering, Jagiellonian University, ul. Granostajowa 7, 30-387 Kraków

Background: Heme oxygenase-1 (HO-1) is a stress-in-ducible enzyme generating iron, biliverdin and carbonmonoxide (CO) from the substrate heme. Enhancementof HO-1 activity, HO-1 overexpression or treatmentwith CO increased VEGF synthesis (Dulak et al.,Antioxid Redox Signal, 2002; 4: 229–240; Józkowicz etal., Antioxid Redox Signal, 2002; 4: 577–585). How-ever, very high activity of HO-1 can generate largeamounts of reactive iron, which decrease VEGF pro-duction. Therefore, here we examined whether the in-fluence of HO-1 on VEGF synthesis is dependent on thelevel of HO-1 expression. Methods and Results: NIH3T3 fibroblasts were engineered to overexpress tTA

regulator and afterwards the cells were transfectedwith pBIL-HO1 plasmid harbouring luciferase and ratHO-1 gene driven by TRE sequence. In the absence ofdoxycycline tTA binds to TRE and activates the expres-sion of luciferase and HO-1. HO-1 expression, as re-flected by enhanced luciferase activity was augmentedwith decreasing doses of doxycycline. Accordingly,VEGF production determined by ELISA was increasedup to 175% of control when HO-1 activity was moder-ately enhanced. Overexpression of HO-1 strongly in-creased a full-length VEGF promoter activity as well asAP-1 activity, as determined in a reporter-gene assay,suggesting the regulation of VEGF synthesis at the


transcriptional level. Interestingly, higher activity ofHO-1 decreased VEGF synthesis, and cells demonstrat-ing highest HO-1 expression generated also more reac-tive oxygen species (ROS), as determined by enhanced2’,7’–dichlorofluorescein fluorescence.

Conclusions: Activity of HO-1 may modulate synthe-sis of VEGF in different way dependently on the level of

HO-1 expression. Thus, moderate HO-1 activity can en-hance VEGF production. In contrast, very high activityof HO-1, potentially associated with high production ofiron and ROS, can inhibit synthesis of VEGF.Supported by grants 3 PO4A 049 22 and 6 P04B 013 from the

State Committee for Scientific Research (KBN).

143 Poster

Functional aspects of prolonged radical scavenging in hypertensive (SHR) rats

Tomasz Wierzba1, Wies³aw Zió³kowski2, £ukasz Nowakowski1, Tomasz Borkowski1, Jan Kaczor2, JerzyPopinigis2

1 — Department of Physiology, Medical University of Gdañsk, ul. Dêbinki 1, Gdañsk, 2 — Department of Bioenergetics, JdrzejŒniadecki University School of Physical Education and Sport, ul. Wiejska 1, 80-336 Gdañsk

Background: Pathogenesis of hypertension involvesreduced microvascular density and inadequate tissueoxygen supply. This promotes excessive generation ofreactive oxygen species (ROS) oxidative damage.

Aim: The study was focused on the effect of anitroxide radical scavenger tempol on: basalhemodynamics, endurance capacity (EC), and mito-chondrial function of hearts taken from spontaneouslyhypertensive rats (SHR).

Methods: Male (250–300 g) SHR rats (N=16) andtheir normotensive control (WKY, N=18) were giventempol (2,2,6,6-tetramethylpiperidine-N-oxyl, 200mmol, 42 days in drinking water), or treated with vehi-cle. Systolic blood pressure (SBP) and heart rate (HR)was measured with a tail-cuff method. EC was obtainedby the time of running to exhaustion. Mitochondrialrespiration rate (MRR) was assayed with a Clark elec-trode. ROS generation was estimated by DCF method(chemiluminescence). Citric syntase and mitochondrialwas assayed with a spectrophotometric assay. SHgroups were assessed within the mitochondria.

Results: In 7-week observation SBP increased in bothWKY rats (from 136 to 145 mm Hg) and in SHR (from193 to 215 mm Hg). It was prevented by tempol (�SBP:–5 and –4 mm Hg, respectively), in parallel to a reduc-

tion in HR (–18, and –38 beats/min). Tempol improvedEC in WKY (by 12%), but changed it for worse (–22%)in SHR. MRR (state 3 and 4) calculated per mg of mito-chondrial protein was faster in SHR than WKY hearts,but not if calculated per mg tissue. RCI was muchhigher in SHR than WKY and was improved by tempol.When succinate was used as a substrate, MRR was re-duced in SHR hearts. Tempol moderately reducedMRR in SHR only. In case of pyruvate and malate em-ployed, it improved MRR in WKY, but worsened inSHR. The latter was spectacular if calculated per mgtissue. Tempol increased ROS generation in WKY, anddecreased it in SHR. SH groups were higher in SHR, es-pecially those treated with tempol. 3-Maleimido-proxylESR assay revealed that tempol may preserveaccesibillity of protein SH group in SHR.

Summary: Elimination of ROS by use of tempolevoked moderate hypotensive effect related tocardioinhibition in both normotensive and SHR rats,but in the latter rats it failed to improve EC. Lack of di-rect functional beneficial effects of tempol in SHR mayresult from the inhibition of mitochondrial respiration.On the other hand, tempol prevented mitochondriafrom excessive ROS formation and at least in part fromoxidative damage of protein SH groups.

144 Poster

Contribution of NADP+-dependent enzymes to regulation of the intracellular

glutathione redox state in rabbit renal tubules

Katarzyna Winiarska1, Micha³ Wêgrzynowicz1, Tomasz Fr¹czyk2, Jadwiga Bry³a1

1 — Zak³ad Regulacji Metabolizmu, Wydzia³ Biologii, Uniwersytet Warszawski, ul. Miecznikowa 1, 02-096 Warszawa,2 — Zak³ad Regulacji Metabolizmu, Instytut Biochemii, Uniwersytet Warszawski, ul. Miecznikowa 1, 02-096 Warszawa

According to our previous findings there is a relation-ship between the rate of gluconeogenesis and the

intracellular glutathione redox state in rabbit kidneycortex tubules. The suggested mechanism of this phe-


nomenon involves the increase in the intracellularNADPH/NADP+ ratio. NADPH, required for GSH re-generation via glutathione redox cycle, can be producedin reactions catalyzed by glucose-6-phosphate dehy-drogenase, isocitrate dehydrogenase and/or malic en-zyme. Thus, the aim of the present study is to deter-mine which of these enzymes are responsible for main-taining the high intracellular GSH/GSSG ratio in rab-bit kidney-cortex tubules.

All three NADP+–dependent enzymes are present inrabbit kidney-cortex tubules. However, the activity ofmalic enzyme (0.87 ± 0.08 nmol x min–1 x mg–1 protein at25oC) is much lower than the activities of the two otherenzymes (23.5 ± 2.5 and 67.4 ± 6.4 nmol x min–1 x mg–1

protein at 25oC for glucose-6-phosphate dehydrogenaseand isocitrate dehydrogenase, respectively). Glu-cose-6-phosphate dehydrogenase and malic enzyme are lo-calized exclusively in cytosol and in mitochondria, respec-tively, while isocitrate dehydrogenase is present in both

compartments. Km values for substrates of the particularenzymes are: glucose-6-phosphate dehydrogenase, 19.5 ±2.8 �M; malic enzyme, 1.3 ± 0.3 mM; cytosolic isocitratedehydrogenase, 12.2 ± 0.5 �M, and mitochondrial iso-citrate dehydrogenase, 3.4 ± 0.2 �M. As the intracellularconcentrations of both glucose-6-phosphate and isocitrateare close to the corresponding Km values, it seems likelythat gluconeogenesis-induced increase in the intracellularconcentrations of these metabolites may affect the activi-ties of glucose-6-phosphate and isocitrate dehydro-genases. Under both gluconeogenic and non-gluco-neogenic conditions, the intracellular concentration ofmalate is far below the Km value for malic enzyme.

In view of these data, reactions catalyzed by both glu-cose-6-phosphate and isocitrate dehydrogenases mightbe considered as main sources of NADPH for maintain-ing high intracellular GSH/GSSG ratio in rabbit kid-ney-cortex tubules.This investigation was supported by the departmental grant.

145 Poster

Selenoenzymes in different types of thyroid tumours

Pawe³ Zagrodzki1, Fergus Nicol2, John Arthur2, Marian S³owiaczek3, Agnieszka Topolska1

1 — Department of Food Chemistry and Nutrition, Collegium Medicum Jagiellonian University, ul. Medyczna 9, 30-688 Kraków,Poland, 2 — Division of Cellular Integrity, Rowett Research Institute, AB21 9SB Bucksburn, Aberdeen, UK, 3 — ThirdDepartment of General Surgery, Collegium Medicum Jagiellonian University, Kraków, Poland

The present study was performed to investigateselenoenzyme activities in thyroid tissues, with refer-ence to other parameters routinely used to characterisethyroid function.

46 samples, obtained during surgical treatment of pa-tients with different types of thyroid tumours, were di-agnosed histologically as 6 groups: (1) nodular goitre(n=20); (2) Graves’ disease, toxic nodular goitre (n=8);(3) follicular or macrofollicular adenoma, (n=5); (4)papillary carcinoma (n=8); (5) medullary carcinoma(n=3), (6) follicular carcinoma (n=2). The followingselenoenzyme activities were measured: cytosolicglutathione peroxidase (cGSHPx), type I and type IIiodothyronine deiodinases (ID-I, ID-II), andthioredoxin reductase one (TrxR) as the biochemicalmarkers for selenium status of the thyroid gland. For33 patients the set of data was completed by determina-tion of serum TSH, fT4, T3, fT3, anti-TG, anti-TPO, andcalcitonin.

The tissues of patients with diagnosis (2) had higheractivities of both ID-I and ID-II when compared with di-

agnosis (4). No other significant difference was foundfor any selenoenzyme. In this context ID-I and ID-IIseem to be the most promising targets for diagnosesand therapy of thyroid tumours [1,2].

In the whole study group statistically significant cor-relation were found between ID-I and ID-II (R=0.746,p<0.0001), whereas cGSHPx activity correlated signifi-cantly with the TrxR activity (R=0.669, p=0.012) incombined groups (4), (5), and (6). These findings are es-sentially in accordance with our previously reporteddata [3]. Additionally, cGSHPx correlated with TSH(R=0.466, p=0.008) and ID-II correlated with fT4(R=0.416, p=0.016). More thorough analysis of datastructure was performed by means of pattern recogni-tion methods.References1. Köhrle J, et al. (1993) Exp Clin Endocrinol, 101: 60–72.2. Wroc³awska M, et al. (1995) Endokrynol Pol, 46: 143–149.3. Zagrodzki P, et al. (2001) Biofactors, 14: 223–227.


146 Poster

Aldehydic lipid peroxidation products in glial tumors

Alicja Zajdel1, Adam Wilczok1, Jerzy S³owiñski2, Krystyna Stêpieñ1

1 — Department of Molecular Biology, Biochemistry and Biopharmacy, Medical University of Silesia, ul. Narcyzów 1,41-200 Sosnowiec, 2 — Department of Neurosurgery and Neurotraumatology, Medical University of Silesia, Bytom

Aldehydic lipid peroxidation products, due to theirhigh reactivity, can initiate the processes of spontane-ous mutagenesis and carcinogenesis and have a signifi-cant role in the aetiology of a number of human dis-eases. Experimental and clinical evidence suggest thataldehydes can also act as bioactive molecules under ei-ther physiological or pathological conditions. These al-dehydic compounds can affect and modulate, at verylow non toxic concentrations, several cell functions, in-cluding signal transduction, gene expression, cell pro-liferation and differentiation, and more generally, theresponse of the target cell.

The brain is vulnerable to oxidative damage. Data per-taining to the amounts of different aldehydes in braintumors are limited. This prompted us to investigate therelationship between the levels of particular aldehydesand the grade of glial tumors.

After tumoral resection the diagnosis was confirmedby histological examination. Secondary lipid pero-

xidation products have been detected as their2,4-dinitrophenylhydrazone (DNP) derivates in speci-mens of glioma collected from six patients withglioblastoma, five patients with astrocytoma grade IIIand four patients with astrocytoma grade II, usingHPLC-MS. A gradient mixture of water, methanol andacetonitrile (acidified with formic acid) and RP C-18column were used for HPLC separations while massspectrometer worked in the negative ionization mode.

The highest amounts of DNP-aldehydes were ob-served in glioblastoma (0.11 ± 0.095 �mol/g).Astrocytoma grade III and astrocytoma grade II con-tained 0.075 ± 0.055 �mol/g and 0.047 ± 0.046 �mol/gof these compounds. ANOVA and Tukey’s HSD testsconfirmed relationship between increased levels ofheksenal, dodecadienal, nonanal, hydroxybutenal,hydroxyundecenal and hydroxydodecenal, decreasedlevels of dihydroksydodecenal and hydroxyoctenal andthe grade of glioma.

147 Poster

Biochemical and histological estimation of experimental hypercholesterolaemia inrabbits as modified by the diet

Jolanta Zalejska-Fiolka, Ewa Birkner, S³awomir Kasperczyk, Aleksandra Kasperczyk, Anna Schneider,Beata Birkner

Katedra i Zak³ad Biochemii Ogólnej w Zabrzu, Œl¹ska Akademia Medyczna w Katowicach, ul. Jordana 19, 41-808 Zabrze

Due to the fact that atheriosclerosis and its complica-tions are the main cause of death in high industrializedcountries, studies of the influence of external factors(especially diet) on the development of atheroscleroticchanges are important. Many reports suggest that eat-ing foods prepared by frying in oils or animal fat mayconstitute health risk. The aim of the study was to com-pare whether oxidized oils and cholesterol can inducehypercholesterolaemia and how different diet influencethe atherosclerosis risk factors and histological profile.

The study was performed on 18 male New Zeland rab-bits (3000 ± 100 g). The animals were randomised into3 dietary groups (n=6). Rabbits received 200 g of stan-dard rabbit diet or different fat composition (200 g/day) as follows: Group I (Control) received standardfodder; Group II (Cholesterol group) received standardfodder with cholesterol (0.5 g/200 g feed); Group III re-

ceived standard fodder with 12 g /200 g feed of oxi-dized rapeseed oil.

The experiment lasted for 24 weeks. At the begin-ning of the experiment and every 6 weeks blood wascollected into heparinized tubes and plasma was im-mediately separated. After 24 weeks the animals wereanesthetized with sodium pentobarbital (60 mg/kg,i.v.) and aortas and liver were quickly removed. Liverand aortas were examined histopathologically. Con-centration of total cholesterol (CH) (enzymaticmethod by Alpha Diagnostics, Germany),HDL-cholesterol (enzymatic method by Alpha Diag-nostics, Germany), LDL-cholesterol (enzymaticmethod by bioMerieux, France) and triglicerydes (en-zymatic method by Alpha Diagnostics, Germany) weredetermined in blood plasma. The results are summa-rized in the table.


The obtained results indicate that: 1) Intensity ofatherosclerotic changes (assessed both biochemicallyand histologically) depended on the diet. Biochemicaland histological changes were significantly larger in an-imals receiving standard fodder with cholesterol thanoxidized rapeseed oil; 2) Administration of oxidized

rapeseed oil did not influence plasma lipids concentra-tion; however, histological investigations indicatedatherosclerotic changes in aortas; 3) Increment of thebody mass of animals receiving standard fodder with12 g oxidized rapeseed oil was bigger than of animalsreceiving cholesterol with standard fodder.

148 Poster

Oxidative stress dependent antiproliferative activity of 2-methoxyestradiol

Agata Zauszkiewicz1, Emilia Syta1, Aleksandra Gil1, Jakub Kêdzior1, Jan Spodnik2, Micha³ WoŸniak1

1 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk, 2 — Katedra Anatomiii Neurobiologii, Akademia Medyczna w Gdañsku, ul. Dêbinki1, 80-211 Gdañsk

2–Methoxyestradiol (2ME) has a cytotoxic effect onvarious proliferating cells. 2ME is also known as an in-hibitor of microtubule dynamics followed by apoptosis.The exact mechanism by which 2ME perturbs targetcell proliferation is still unclear.

We have demonstrated for the first time thatestradiol metabolite — 2-methoxyestradiol is a powerfulinducer of oxidative stress in osteosarcoma 143B cell

line. Oxidative stress in cells exposed to 2ME was evi-dent before inducing pleiotropic mechanism involvingcell cycle arrest at G1 and G2/M phase respectively.The ability of 2ME to inhibit cell cycle at the respectivepoint was found to be concentration dependent. Thepresent data suggest oxidative stress dependent mech-anism of apoptosis induction by 2ME at cellular level.

149 Poster

Modulatory effects of quercetin and rutin on neutrophil apoptosis

Ma³gorzata Zieliñska

Katedra Biochemii Farmaceutycznej, Akademia Medyczna im. K. Marcinkowskiego, ul. Grunwaldzka 6, 60-780 Poznañ

Neutrophils play a primary role in the initiation andpropagation of inflammatory responses. Theirapoptosis is a major mechanism associated with theresolution of inflammatory reactions. Caspase-3 acti-vation is the first step in the execution phase ofapoptosis. Human neutrophils were isolated and cul-

tured for up to 24 hours. PMA (200 nmol/L)-treatedneutrophils were assessed for caspase activity in vitroand were found to yield no detectable DEVD-AMCcleavage activity at any time point tested. However,when cells were pre-treated with diphenyleneiodonium (DPI, 10 micromol/L), an inhibitor of the


*p= 0.05; **p= 0.01

NADPH oxidase, there was a time-dependent induc-tion of caspase activity. These findings suggest a spe-cialised caspase-independent pathway of cell death inactivated neutrophils. When cells were cultured with1–100 �M quercetin or rutin for 24 h, caspase-3 wasalso activated. Induction of apoptosis by quercetin

and rutin was accompanied by a decrease of the gener-ation of reactive oxygen species (ROS) in freshly iso-lated cells.

Regulation of caspase activity by flavonoids may rep-resent a potent mechanism triggering apoptosis and re-solving inflammatory disorders.

150 Poster

Effect of flavonoid glycosides on angiotensin II induced changes in redox status inPC12 cells

Bogdan Zieliñski1, Izabela Berdowska1, Ewa Seweryn1, Izabela Fecka2

1 — Katedra i Zak³ad Biochemii Lekarskiej, Akademia Medyczna we Wroc³awiu, ul. Cha³ubiñskiego 10, 50-368 Wroc³aw,2 — Katedra i Zak³ad Farmakognozji, Akademia Medyczna we Wroc³awiu, Wroc³aw

Cellular redox status plays a modulatory role in signaltransduction. Luteolin-7-O-rutinoside (lutA) anderiodictiol-7-O-rutinoside (erioB) having antioxidativeproperties were chosen to study their influence on re-dox balance processes triggered by angiotensin II(AngII) through AT2 receptor in rat pheo-chromocytoma cell line PC12. PC12 cells werepreincubated with Losartan and either lutA or erioBfor 10 minutes, followed by incubation with AngII for0.5, 2 and 5 minutes. For comparison, PC12 cells wereincubated with each of these substances alone. Subse-quently, the cells were lysed and glutathione

peroxidase (GPx) activity was measured. After2-minute AngII stimulation GPx activity increased bynearly 40% in comparison to 0.5-minute stimulation.This effect was abolished by lutA, whereas prein-cubation with erioB enhanced GPx activity by 40% after0.5-minute AngII stimulation, and lowered GPx activityby 34% after 5-minute AngII stimulation as comparedwith AngII alone. It might be suggested that AngIIthrough its AT2 receptor in PC12 cells induces increasein hydrogen peroxide (GPx substrate) concentrationfollowed by GPx activity increase. This effect was mod-ulated by flavonoid glycosides lutA and erioB.

151 Poster

Peroxidation of low-density lipoprotein lipids induced by interaction with glycatedhemoglobin and hydrogen peroxide

Jolanta Zuwa³a-Jagie³³o, Anna Koœæ

Katedra Biochemii Farmaceutycznej, Akademia Medyczna, ul. Szewska 38, 50-139 Wroc³aw

Hyperglycemia is considered a key causal factor inthe development of diabetic vascular complications andcan mediate its adverse effects through multiple path-ways. There are several equally defensible hypotheseson the roots of complications including, but not limitedto, the early glycation end products (e.g. glycated hemo-globin, GHb), oxidative stress, and altered lipoproteinmetabolism.

In the current study we focused on the final productsof low-density lipoprotein (LDL) peroxidation (thetiobarbituric-acid reactive substances, TBARS) in-duced by GHb and free hemoglobin (Hb). We demon-strate that although Hb is capable of inducing LDL lipidoxidation, the peroxidative reactivity of GHb somewhatexceeds that of Hb.

Leakage of Hb from damaged red blood cells in many in-cidents at vascular sites of high shear forces (blood vessel

bifurcations) or in intramural hemorrhages may not besufficient to induce LDL lipid oxidation. However, our re-sults indicate that in the presence of minor amounts (10�M) of hydrogen peroxide, even in the absence of free ox-ygen, Hb and GHb become active promoters of LDLperoxidation.

In the patient with diabetes, already burdened withincreased oxidative stress from hyperglycemia, abilityof haptoglobin (the endogenous antioxidant) to sieveinto the vessel wall to neutralize Hb or GHb is likely tobe of great importance. The stoichiometries ofhaptoglobin 1-1 and 2-2 binding to hemoglobin are iden-tical. Our results showing a difference between theamount of GHb-inducible oxidation between our twodifferent preparations was not caused by a systematicerror in the determination of the glycated hemoglo-bin-binding capacity of our preparations of two


haptoglobin types. At a concentration of 10 �M GHband 5 �M haptoglobin (concentrations at which wefound haptoglobin 1-1 was significantly superior tohaptoglobin 2-2 in protecting against GHb-inducedLDL oxidation), we found that there was no significantdifference between the amount of GHb using ourhaptoglobin 1-1 and our haptoglobin 2-2. Therefore, theinferior antioxidant capacity of our haptoglobin 2-2could not be explained by a lower effective GHb-bindingcapacity than our haptoglobin 1-1.

Our studies suggest that GHb and Hb are similar intheir ability to induce LDL peroxidation. The differencebetween haptoglobin 1-1 and 2-2 in inhibiting the oxida-tion of LDL may be the result of differences in the abil-ity of the different types of haptoglobin to prevent therelease of heme. High reactivity of glycated hemoglobintoward LDL would indicate its potential as anatherogenic factor.

152 Poster

A new method of oxidative stress intensity evaluation in children with juvenilechronic arthritis

Dorota ¯urawa1, Joanna Renke2, Stefan Popadiuk2, Maria Korzon2, Joanna Skórko-Glonek1, BarbaraLipiñska1, Bo¿ena Bugajczyk3, Micha³ WoŸniak4

1 — Katedra Biochemii, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk, 2 — Klinika Pediatrii, Gastroenterologii i OnkologiiDzieciêcej, Akademia Medyczna w Gdañsku, ul. Nowe Ogrody 1-6, 80-803 Gdañsk, 3 — Oddzia³ Dzieciêcy, Szpital Reumatologiczny,Sopot, 4 — Katedra i Zak³ad Chemii Medycznej, Akademia Medyczna w Gdañsku, ul. Dêbinki 1, 80-211 Gdañsk

Juvenile chronic arthritis (JCA) is a group of systemicinflammatory disorders with features of autoimmunedisorder, affecting children. The clinical picture of theJCA is formed by the inflammatory changes in connec-tive tissue: its cells, basal substance, and collagenfibres, resulting in the inflammation of joints.

It is well known that reactive oxygen species (ROS) aregenerated during chronic inflammatory process. Overpro-duction of ROS results in prooxidant–antioxidant bal-ance disturbances and induces an increased damage to allcellular macromolecules: proteins, DNA, lipids. The ac-tion of ROS on proteins results in the formation of car-bonyl groups that are believed as a respected marker offree radical reactivity [1].

The evidence that oxidative damage occurs in rheu-matoid diseases, among them in JCA, is very strong [2].The increase of plasma protein oxidation products, ex-pressed by the level of carbonyls, was recently evalu-ated in a group of children with JCA [3].

Objective of the study was to find in which fraction ofplasma proteins in children with JCA the oxidativedamage is the highest and if it correlates with any clini-cal or laboratory parameters.

Oxidative damage was monitored by assay of proteincarbonyl group content [4]. To assay the carbonyl groupsin plasma proteins fractions, a new method, based on elec-trophoresis and detection of the carbonyl derivatives(dinitrophenylhydrazones) by immunoblotting was de-vised.

The results of the preliminary study showed that it isalbumin and gamma-globulines that carry the main at-tack of ROS and are oxidatively modified by them. Theresults showed also the strong prevalence ofgamma-globulin destruction in a group of children withsystemic onset JCA and more often in children withpolyarthritis and oligoarthritis. In other fractions ofplasma proteins carbonyls were detected in three cases.The oxidative modification concerned alpha-2- andbeta-globulines, and was not intense.References1. Halliwell B, Gutteridge JMC (1990) Free Radicals in Biology and

Medicine, Oxford Science Publications, Third Edition.2. Garibaldi S, Aragno I, Odetti P, Marinari UM (1994) Biochem

Molec Biol Int, 34: 729–36.3. Renke J, Popadiuk S, Korzon M, Bugajczyk B, WoŸniak M (2000)

Free Radic Biol Med, 29: 101–104.4. Levine RL, Williams JA, Stadtman ER, Shacker E (1994) Meth in

Enzymol, 233: 346–363.


153 Poster

Oxidative stress and magnesium sulfate — implication for therapeutic treatment

Ludmi³a ¯yliñska1, Ewa Gulczyñska2

1 — Instytut Fizjologii i Biochemii, Zak³ad Biochemii Lekarskiej, Uniwersytet Medyczny w £odzi, ul. Mazowiecka 6/8, 92-215£ódŸ, 2 — Klinika Neonatologii, Instytut, £ódŸ

Magnesium sulfate as a calcium antagonist is themost widely used parental drug for tocolysis. Data fromcase-control studies have suggested that in utero expo-sure to MgSO4 may protect very low birth-weight new-borns from cerebral palsy. Moreover, the pretreatmentwith magnesium could have more beneficial effectsagainst hypoxic/ischemic brain damage blocking theNMDA receptor and hindering calcium influx into theneurons. Mg2+supplementation may also reverse theharmful effects of oxygen radicals, excitatory amino ac-ids and tromboxane A2.

We have previously shown that several erythrocytemembrane components were changed in asphyxiatednewborns. We observed increased peroxidation ofmembrane lipids, a lowered amount of band 3, and al-tered activities of the membrane enzymes(serine/threonine kinases and tyrosine kinases) andplasma membrane calcium pump. These changes couldbe a result of the action of reactive oxygen species. Thepresent study was undertaken to characterize selectedmembrane components in erythrocytes of preterm neo-nates whose mothers received MgSO4 before delivery.Control group consisted of simultaneously born infantsof similar gestational age. We have determined the ac-

tivities of Mg2+-ATPase and Mg2+-dependentCa2+-ATPase, and the level of anion-exchange proteinusing monoclonal antibodies, in the both groups of in-fants.

These parameters were examined directly after thedelivery and 24 hours later. We have observed no dif-ference in the activity of Mg2+-ATPase in both groupsafter 24 h. The basal activity of calcium pump in thecontrol group was 1.5–2 times higher than that ofMgSO4–treated one (3.92 ± 0.47 vs. 1.94 ± 0.63micromoles/mg protein per h). The ability of the pumpfor stimulation by the physiological activator,calmodulin, was similar after delivery, but increased by50% in Mg2+-treated newborns after 24 hours. More-over, in this group the amount of anion-exchange pro-tein was nearly two times higher than that of controlneonates (373 vs. 243 OD/�g of protein).

Our results suggest that magnesium sulfate treat-ment could be a factor that limits the second wave ofcell membrane damage after delivery.Supported in part by the grants No. 5P05E 09224 from the

State Committee for Scientific Research (KBN), andNo. 503 from the Medical University of Lodz.


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