P74 PROLACTIN-NEW TUMOR MARKER FOR BREAST CANCER?

Zlata Mujagic, Hamza Mujagic

Purpose:The aim of this study was to assess the usefulness of prolactin as a potentially useful prognostic tool in breast cancer patients.

Methods: The main experimental group consisted of 47 female patients with histologically confirmed diagnosis of breast cancer. The results were compared with prolactin levels of apparently clinically healthy women, and female patients with other locations of cancer. Results were processed by means of t-test, two way analysis of variance, and logistic linear correlation model.

Results: The circulating levels of prolactin before treatment as well as their frequencies were significantly higher in breast cancer patients in comparison to controls. The average prolactin concentration in patients with metastatic disease was significantly higher than in those without detectable metastases. There also was a significant negative correlation between prolactin levels and time intervals before the occurrence of metastases in all, and especially in hyperprolactinemic patients. Of special interest is to mention the existence of highly significant negative correlation between prolactin levels after treatment and the results of treatment.

Conclusions: These results suggest that prolactin may be a useful prognostic tool in breast cancer patients, but larger series of patients are necessary to be included before final judgment can be made.

P75

ASSOCIATION OF K-ras MUTATIONS WITH p16^INK4A AND MGMT METHYLATION IN HUMAN COLORECTAL CANCER

Milena DRAGIĆ¹, Slavica KNEŽEVIĆ-UŠAJ², Bogomir DIMITRIJEVIĆ¹, Koviljka KRTOLICA¹

Aberrant DNA methylation has been identified as an important epigenetic mechanism for inactivation of tumor suppressor genes and DNA repair genes such as p16^INK4A and MGMT respectively, during colorectal carcinogenesis. We examined the methylation status of CpG islands in promotor region of p16^INK4A and MGMT gene in selected group of 37 patiens, with diagnosis of colorectal carcinoma and compared obtained results with presence of mutations in K-ras gene for the same tumor samples, in order to evaluate the possible association between this genetic event and aberrant methylation of p16^INK4A and MGMT gene, respectively. DNA was extracted from paraffin-embedded tissue semples, using standard protocol involving proteinase K digestion, phenol/chloroform/isoamil extraction and ethanol precipitation. DNA methylation patterns were determined by chemical bisulphite modification of unmethylated, but not the methylated cytosines to uracil and subsequent PCR, using primers specific for either methylated or the modified unmethylated DNA. K-ras mutations were present in 51.4% (18 of 35) studied samples. Methylated p16^INK4A was found in 54.1% (20 of 37) , and methylated MGMT gene in 45.9 % (17 of 37) of samples. Among 18 tumors with K-ras mutations, p16^INK4A methylation was detected in 10 (55.6%), and MGMT methylation in 8 (44.4%) of samples. Six of 7 (85.7 %) tumors with G to A mutation in K-ras showed MGMT methylation, whereas only 2 of 11 (18.2 %) of the tumors with other kind of K-ras mutations were methylated. This demonstrate a clear association between inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras gene. Our results suggest that p16^INK4A and MGMT methylation occurs frequently in human colorectal cancers and is closely associated with K-ras mutations. These associations indicate that aberrant methylation have important interactions with genetic lesions in pathogenesis of this cancer type.

Аnica I. HORVAT, Snježana B. PETROVIĆ, Nataša A. TERZIĆ, Miroslav A. DEMAJO

The aim of this work is to study the modulation of Na,K-ATPase and Mg-ATPase activities from rat brain nerve terminals after irradiation with -rays from a ⁶⁰Co source. Ionizing irradiation is widely used for diagnostic and therapeutic purposes. Despite extensive studies of ionizing radiation effects on various tissues, there is lack of information concerning brain irradiation effects. With the aim to explore early (60 min) neuromodulatory effect of -rays, we measured activities of synaptosomal Na,K-ATPase and Mg-ATPase of wholebody acute irradiated rats (9.6 Gy, 10.7 cGy/min). Female cycling (CY) and bilateraly chronically ovariectomized (OVX) rats were divided into three groups: the control group (C) were under physiological conditions, animals whole body irradiated (9,6 Gy, 10,7 cGy/min) were termed as the irradiated group (IR). During irradiation, the animals were kept in plywood boxes. Because of the immobilization stress as a positive control third group, the animals were treated as the irradiated group but without being irradiated (IM). One hour after irradiation, membranes of nerve endings (SPM) were isolated from whole brains and the activities of ATPases were determined under in vitro conditions. Na,K-ATPase and Mg-ATPase acivity were significantly higher in CY compareed to OVX. In IM the activities of both enzymes were higher than in C of CY, but deprivation of ovarian hormones supress immobilization induced increase of Na,K-ATPase. One hour after irradiation, in CY activities of Na,K-ATPase and Mg-ATPase decrease in the respect to activity of IM as well as Mg-ATPase of OVX rats, while Na,K-ATPase was increased. It is seem that ovarian hormones modulate stress-and irradiation-induced response of Na,K-ATPase, while hormonal status does not influence neither stress nor irradiation effect on Mg-ATPase activity. It is obvious that single whole body irradiation after one hour inhibits stress-induced increased Na,K- and Mg-ATPase activity nearly reaching the control level.

V.Arsova, J Lavcanska

Introduction: Elevated lipoprotein(a) and homocystein levels have been described as independent risk factors for coronary artery disease and venous thrombosis. Statins (simvastatin) are a major advance in the treatment of hypercholesterolemia because they act as inhibitors HMG, CoA-reductase. The aim of the study was to evaluate the potential relationship of lipoprotein(a) and serum levels before and after 12 months simvastatin therapy in hypercholesterolemic patients with acute myocardial infarction. Subjects and methods: The study group of 65 patients (average age 52±14 years, 45 males, 20 females) were monitored at baseline and 12 times within 12 months, measurement of:lipoprotein(a) cut-off <30mg/dl, and total chomocystein, tCHy cut-off < 10mmol/l. Serum levels of Lp(a) were measured by immoturbidimetric method on Cobas Mima (Roche); tHCy by Abbott AYSYM assay. The doses of simvastatin therapy were 40mg and 20mg depending of cholesterol levels. Results: According to our results there were not statistically significant differences at baseline and after therapy; Lp(a) mg/dl (104±82 v.s 99.86±83.5) p>0.05;tHCYmmol/l (15.8±3.23 v.s 14.72±3.11) p>0.05. There was statistically high correlation between Lp(a) concentration at baseline and 12 months later r=0.936 p<0.0001 and of tHCy r=0.896 p<0.001. All patients had the same Lp(a) levels at baseline and 12 months follow up, more over, all the patients had approximately the same tHCy values at baseline and 12 months follow up. Conclusion: Significant reduction of total and HDLc represents good results of therapy, tHCY levels can easily be treated with vitamin supplements, while Lp(a) levels are stable over time.

Siarhei V. LUPACHYK, Zoya V. NIATSETSKAYA, Liliya I. NADOLNIK

It's defined that high iodine doses (HID) induced thyrocyte apoptosis and possibly explained by oxidative stress activation. We aimed investigate HID influence on thyroid functional activity and its pro/antioxidant status.

All experiments were performed on female Wistar rats (180-200 g, n=10) and treated with KI 0.7, 7 and 70 mg/kg weight (10, 100 and 1000 physiological doses, respectively and sacrificed after 24 hours). As a control we used animals receiving daily physiological iodine dose (0.07 mg/kg). Thyroid functional activity and pro/antioxidant status was determined by measuring total, free and protein-binding tissue iodine levels, thyroperoxidase activity and thyroid thiobarbituric acid reactive substances (TBARS) levels, catalase, SOD and glutathione reductase (GR) activities, respectively.

Our data indicate that HID inhibits thyroid functional activity on two levels. Firstly, 1.58- and 1.18-fold total iodine levels (7 and 70 mg/kg, respectively) and 1.30-fold free iodine levels (7 mg/kg) reduction evidences iodine uptake suppression. Secondly, this accompanied by iodine organification inhibition: 3.98-fold thyroperoxidase activity (70 mg/kg) and 1.12-, 2.19- and 1.23-fold reducing the protein-binding iodine levels (0.7, 7 and 70 mg/kg, respectively). Interestingly also that KI 70 mg/kg treatment leads to significant 1.18-fold increasing of thyroid weight. HID treatment caused 1.14-, 1.14- and 1.25-fold tissue TBARS levels and 1.17-, 1.26- and 1.51-fold catalase activity rising in rat thyroids treated with 0.7, 7 and 70 mg/kg, respectively. GR activity was significantly 1.21-fold increased to only KI 70 mg/kg treated rats and SOD activity was not altered. Markedly rising catalase activity may be caused by HID induction H2O2 production by thyrocytes.

Our results suggest that HID, presumable, have a toxic effect on thyroid, since even single KI treatment inhibit thyrocyte functional activity by suppression as iodine uptake as its organification. This accompanied pronounced oxidative stress activation and rising of reactive oxygen species and lipid peroxidation toxic products.

P79

Karine Barsegyan, Sedrak Ghazaryan, Nina Movsesyan, Nina Alchujyan, Hovhannes Movsesyan, Gohar Elbakyan and Guevork Kevorkian

This study was designed to elucidate the association of blood formed elements (BFE) (platelets, neutrophils, monocytes and lymphocytes) and NOS isoenzymes with the pathogenesis of familial Mediterranean fever (FMF). We have synthesized L-arginine analogue with blocked a-NH₂ group (CH1) and examined its effect on the NOS activity in whole blood and BFE of healthy humans (3 women of 38-51 years old) and patients with FMF (3 women of 16-50 years old). The effect of calmodulin (CaM) was also studied. No close relationship was observed between the production of NO²⁺/NO³⁺ and L- citrulline, as well as the consumption of L-arginine in whole blood and BFE. Volunteers’ studies showed the NOS activity was decreased in BFE, especially in platelets and neutrophils, whereas the plasma level of NO²⁺/NO³⁺ was increased for 1,2-1,7 times in patients with FMF, as compared to controls. It appears, the nitrite/nitrate anions caused the feedback inhibition of NOS. The elevated glucose level in plasma and erythrocytes sedimentation rate observed in patients with FMF seemed to be caused by the decrease of NOS activity, since NO/NOS is known regulate the rheological behavior of erythrocytes and partially regulate the concentration of L- arginine, which is in turn involved in the regulation of glucose and insulin levels in human blood. The addition of CH1 in the incubation mixture was mainly accompanied with shift to the Ca²⁺- CaM dependent NOS activity. CH1 increased markedly the dropped NOS activity in platelets and neutrophils of women with FMF. The effects of CaM and CH1 seemed to be different on various NOS isoenzymes and should further be studied.

Ayse G. CANSEVEN¹, Ummühani OZEL² , Ayse BILGIHAN² and Nesrin SEYHAN¹

Myeloperoxidase (MPO), a bactericidal enzyme secreted by activated phagocytes, specifically catalyzes the production of hypochlorous acid (HOCl) from chloride and hydrogen peroxide.

The study was assessed to evaluate the influences of in vivo exposures to 10 G (Gauss) and 30 G of magnetic fields on MPO activities of lung, brain and kidney tissues in guinea pigs. MPO activity was measured as a marker of neutrophil accumulation in those tissues.

Thirthy six male, 250-300 g weighted guinea pigs were used. Twenty eight guinea pigs were exposed to the fields of 50 Hz 10 G and 30 G with the period of 4 hours/day and 8 hours/day for 5 days in 4 different groups. Eight animals were served as control, keeping at the same conditions without being exposed to any magnetic field. Magnetic field was generated by a pair of Helmholtz coils. Circular coils pair of Helmholtz configuration was used. Animals were placed pairly in plastic cages which were positioned at the center of the Helmholtz Coil during exposure conditions, to avoid any distortion of the generated magnetic field. Ambient geomagnetic field was measured as 0.3 G in the laboratory.

Brain, lung and renal tissues were homogenized according to methods of Matsuo Y. et.al., Koike K. et al. and Lopez-Neblina F. et.al. respectively. MPO activities in these tissue samples of exposed and unexposed guinea pigs were determined by measuring the H₂O₂-dependent oxidation of o-dianisidin by the method of Glowick SP. et al. Mann Whitney-U test was applied for statistical analysis.

MPO activities in brain tissues of guinea pigs exposed to the magnetic fields of 10 G and 30 G were found increased with repect to the controls for both of the exposure periods. Increased MPO activities were found in lung tissues under the effect of 10 G magnetic fields for both of the exposure periods whereas decreased MPO activities were determined for the magnetic field of 30 G. The decrease in MPO activities for 4 hours/day of exposure times was found statistically significant (p=0.002). Renal MPO activities were also found increased for both of the magnetic fields and the exposure periods. The increase in MPO activities was found statistically significant (p= 0.004) under the effect of 10 G for 5 days with the exposure period of 4 hours/day.

Ayse G. CANSEVEN¹, Ummühani OZEL² , Ayse BILGIHAN² and Nesrin SEYHAN¹

1. 
   Department of Biophysics, Gazi University, Medical Faculty, Ankara, TURKEY
   
2. 
   Department of Biochemistry, Gazi University, Medical Faculty, Ankara, TURKEY
   

The aim of the present study was to investigate the effect of 50 Hz, 20 G magnetic field on MPO activity in renal tissues of guinea pigs.

Magnetic field was generated by a pair of Helmholtz coils. Circular coils pair of Helmholtz configuration was used in the vertical manner. Twenty two male, 250-300 g weighted guinea pigs were used. Fourteen guinea pigs were exposed to the field of 50 Hz, 20 G with the exposure periods of 4 hours/day and 8 hours/day for 5 days in 2 different groups. Eight animals were served as control, keeping at the same conditions without being exposed to any magnetic fields. Animals were placed pairly in plastic cages which were positioned at the center of the Helmholtz Coil during exposure conditions, to avoid any distortion of the generated magnetic field. The animals were kept in the laboratory at a room temperature of 23°C, a day and night cycle of 12 hours and ambient geomagnetic field of 0.3 G.

MPO activities in renal tissues of exposed and unexposed guinea pigs were determined by measuring the H₂O₂-dependent oxidation of o-dianisidin according to methods of Lopez-Neblina et al. and Glowick SP. et al. Mann Whitney-U test was applied for statistical analysis.

MPO activities in kidney tissues of guinea pigs exposed to the 50 Hz, 20 G magnetic field were found increased in both of the exposure periods. The increases in MPO activities of renal tissues were statistically significant both application times of 4 hours/day (p=0.014) and 8 hours/day (p=0.001). The exposure periods of 8 hours/day was found more effective.

P82

LYSINE-RICH HISTONES AS A MODEL SYSTEM TO INVESTIGATE NONENZYMATIC GLYCOSYLATION IN E. COLI

Georgi STOYNEV, Ljuba SREBREVA, Ivan IVANOV

Objective: Nonenzymatic glycosylation (glycation) is a multystep reaction between free amino groups in the side chains of proteins and various carbonyl compounds. The initially formed unstable Schiff and Amadory products (called early glycation products) are spontaneously converted into more stable terminal adducts -advanced glycation end products (AGEs). For a long time it has been thought that AGEs are typical for the long-lived proteins in higher eukaryotes only. Recently, it has been found in our laboratory that E.coli proteins, including recombinant human interferon gamma (hrIFN-) are also subjected to glycation (Mironova, R. et al, Mol. Microbiol. (2001) 39, 1061-1068). The aim of this study is to develop an assay system for studying the glycating capability of bacterial lysates based on the lysine-rich histones H1 and/or H5 subvariant. Methods: H1 or H5 histones are extracted from rat liver or chicken erythrocytes by perchloric acid, dissolved in water and dialyzed overnight at 4^o C against clear (centrifuged) bacterial lysates. Accumulation of AGEs is monitored by fluorescence (_ex 365 nm and _em 443 nm) and the accompanying changes in the protein structure are monitored by electrophoresis. Results and Conclusion: In kinetic studies we have detected significant accumulation of AGEs in both H1 and H5 histones and also fragmentation and cross-linking. Using known AGE inhibitors (as thiamine, pyridoxine and aspirin) we have registered a detectable decrease in the level of AGEs. Our results showed that aspirin is the most effective inhibitor of glycation. In conclusion, the in vitro incubation of lysine-rich histones with bacterial lysates produces AGEs on the native proteins.

* Snežana RADIVOJŠA, ** Vladislav VARAGIĆ and *** Slobodan MILOVANOVIĆ

The role of nitric oxide in inhibitory non-adrenergic non-cholinergic neurotransmission was studied on longitudinal muscle of the guinea pig ileum. The most widely reported action of sodium nitroprusside (SNP), a nitric oxide donor compound, in the gut is relaxation of smooth muscle. The isolated segments of ileum were maintained in Tyrode solution and the addition of SNP (10^-10–10^-5M) to the organ bath concentration-dependently inhibited contractions, caused by electrical stimulation (8-79%) and acetylcholine (24-62%). Segments of ileum were exposed to 1M SNP during 60min to induce in vitro tolerance to SNP and then exposed to increased concentrations of SNP. The degree of relaxation of contraction caused by electrical stimulation and acetylcholine was 4-50% and 6-30%, respectively. Among the most popular theories for nitrate tolerance is the “intracellular sulfhydryl depletion hypothesis”. The influence of the N-acetylcysteine, donor of sulfhydryl group, on tolerance caused by SNP was investigated. Our results showed that N-acetylcysteine (1mM) can change the activity of SNP (electrical stimulation – 8-63%) and it was found that exogenously added thiols can partially reverse nitrate tolerance.

Higher concentrations of SNP induced a biphasic response, an immediate relaxation (1-3min) followed by prolonged contraction (10min). The relaxations evoked by NO liberated from SNP were blocked 20% by methylene blue (10mM). The results suggest that relaxant responses to exogenous nitric oxide in guinea pig ileum are mediated via the activation of soluble guanylate cyclase and the formation of guanosine-3’,5’-cyclic monophosphate. The contractions evoked by SNP were inhibited 40% by atropine (1M). Concerning the obtained results it can be concluded that contraction evoked by SNP on smooth muscle cells are also mediated by activating acetylcholine release from neurons.

P84

EXAMINATION OF ELECTRIC FIELD EFFECTS ON LIPID PEROXIDATION AND ANTIOXIDANT ENZYMES BY USING EXPERT SYSTEMS

Göknur GÜLER¹ and Fırat HARDALADz

Malondialdehyde (MDA) and superoxide dismutase (SOD) levels in spleen and testis tissues of guinea pigs which were exposed to different periods of Extremely Low Frequency ( ELF ) electric fields were determined and the results are applied to neural networks as learning data and the training of the feed forward neural network is realized. At the end of the training, to determine the effect of the electric field on tissues in computer without applying electric field and without using too many guinea pigs and to form a database for the researchers in this field are aimed.

Five groups of 15 male white guinea pigs (150-200 g) were exposed to 50 Hz 5 kV/m ELF electric fields. Each group was exposed daily for 8 hours for 1 day, 3 days, 5 days, 7 days and 10 days. The 75 guinea pigs were examined according to the exposure periods while 15 guinea pigs, which were not exposed to any electric field, formed the control group.

The effect of 50 Hz electric field exposure on MDA and SOD levels was investigated for different application periods. The increase in MDA and SOD levels of spleen and testis tissues was found to depend significantly on the type of electric field and the length of exposure.

After the experiments, the prediction of the neural network is averagely 99 %. Those percentiles of the prediction performance of the neural network belonging to experiment results of electric field were so high; this fact shows that the feed forward neural networks which are used many fields could be applied in the studies of electric field too. Furthermore this study may form a database for the scientists investigating the effects of electric fields on lipid peroxidation and antioxidant enzymes.