EFFECTS OF SUBCHRONİC TREATMENT OF THİOCARBAMİDE ON HAEMATOLOGİCAL AND BİOCHEMİCAL CONSTİTUENTS OF RATS

EFFECTS OF SUBCHRONİC TREATMENT OF THİOCARBAMİDE ON HAEMATOLOGİCAL AND BİOCHEMİCAL CONSTİTUENTS OF RATS

İsmail ÇELİK, PhD Hanefi ÖZBEK, MD, PhD * and Yasin TÜLÜCE

The effects of sublethal concentration of Thiocarbamide on various haematological and biochemical costituents of rat was investigated under laboratory conditions. 250-ppm of Thiocarbamide was administered orally to 8 rats ad libitum during the tests for 25 days cosecutively.

Various haematological and biochemical costituents of rat were determined after treatment. According to results, the treatment of Thiocarbamide caused significant increases in lactate dehydrogenase (LDH) and creatine phosphokinase (CPK), while the level of alanine aminotransferase (ALT) was decreased. Aspartate aminotransferase (AST) and amylase did not change. On the other hand, the treatment of Thiocarbamide on rat also resulted in a different effect on the level of blood costituents in comparision to that of control rats. While the levels of white blood corpuscles (WBC) and thrombocyte (PLT) were increased significantly by Thiocarbamide, the other parameters did not change. With regard to the biochemical characteristics, while the level of total cholesterol (TC) and high density lipoproteine cholosterole (HDL-C) were increased significantly by Thiocarbamide, the other consituents did not change. It is concluded from this study, that Thiocarbamide may cause toxicity on different tissues in rats.

P305

INACTIVATION OF UBIQUINOL OXIDASE IN RB. CAPSULATUS AND CHARACTERISTICS OF THE NULL MUTANTS

Özlem AKKAYA¹, Sevnur MANDACI², Fevzi DALDAL³, Yavuz ÖZTÜRK²

Gram-negative facultative photosynthetic bacterium Rhodobacter capsulatus can grow through respiration using two different metabolic pathways that are branched after the quinone pool. In the main respiratory chain, electrons are first transported to cytochrome bc₁ complex, then to cytochrome c₂/c_y and finally to cytochrome cbb₃ oxidase, the last electron acceptor. The other metabolic pathway is an alternative respiration pathway in which electrons are transported from the quinone pool to the ubiquinol oxidase (Q_ox) irrespective of any electron carrier.

In this study, to characterize respiratory function of the novel electron carriers, several chromosomal knockout Q_ox^- mutants which eliminated the alternative respiratory pathway were obtained. To construct ubiquinol oxidase chromosomal knockout mutants, inactivation of cydAB genes encoding ubiquinol oxidase was achieved. Structural cydAB genes were inactivated using a gentamicin marker. Cloning 2.7-kb cydAB::Gm^R fragment from pOZ1 plasmid containing inactive cydAB::Gm^R on pRK vector, yielded pYOZ1. pYOZ1 was conjugally transferred from E. coli strain into the Gen Transfer Agent (GTA) producing R. capsulatus Y262 strain and resulted in pYOZ1/Y262. The cydAB::Gm^R fragment was introduced into the various different electron carrier bearing R. capsulatus strains by homologous recombination via GTA cross, and yielded ubiquinol oxidase chromosomal knockout mutants. It was observed that all of these mutants grew via cytochrome cbb₃ oxidase by respiration, but had some differences in their photosynthetic phenotypes.

The cydAB::Gm^R chromosomal knockout mutants which were obtained from this research are important to determine efficiency of the novel electron carriers during the mitochondrial type respiration. Moreover these mutants will be important to investigate the in vivo function of the ubiquinol oxidase in photosynthesis.

*This study was supported by ICGEB grant (TUR99-01) to S.M.

P306

erythropoIetIn ıncreaseS Neurotrophıc FaCTORS

IN MICROGLIA CELLS

Filiz KURALAY¹ , Basak Bingol¹, Sermin Genc², Kursat GENC³

Microglial cells play an important role in the development of neurons and in the repair processes of the central nervous system (CNS) injuries. Through such growth factors as erythropoietin(EPO) and interferon(IFN) gamma which they produce and release, microglias provide survival support. Some of these growth factors are neurotrophic agents and the best known are neurotrophic factor 3(NT3),neurotrophic factor 4(NT4), and brain derived neurotrophic factor(BDNF). In this study, we aimed to investigate whether IFN+Lipopolysaccharide (LPS) and amyloid beta(AMY) as toxic stimulator agents and also EPO as a neurotrophic agent have an effect on the production of neurotrophic factors. which are BDNF, NT3 and NT4 in microglial cell lines. For obtaining microglial cells, 8 neonatal 0.day BALB/C mouse brains were used. After mechanical separation, polilizin covered culture flasks were used for sowing in DMEM/F12 medium containing 10% fetal bovine serum. At various doses, recombinant mouse EPO, LPS, IFN gamma and AMY beta were added to the culture medium. LPS, IFN gamma and AMY beta were added at the dose of 1 microgram/ml, 100 U/ml and 50 microgram/ml, respectively; while EPO was used at the three different concentration(0.1, 1, and 5 U/ml). No cytokine addition was used for control culture. Experimental research was conducted on 9 groups, involving 3 cell lines in every one, as follows: 1) Control, 2) LPS (1 microgram/ml) + IFN gamma(100 U/ml), 3)AMY (50 microgram/ml), 4) EPO(0.1 U/ml), 5) EPO (1 U/ml), 6) EPO(5 U/ml), 7) LPS (1 microgram/ml) +IFN gamma (100 U/ml) + EPO (0.1 U/ml), 8) LPS (1 microgram/ml) +IFN gamma (100 U/ml) + EPO(1U/ml), 9) LPS (1 microgram/ml) +IFN gamma (100 U/ml) + EPO (5 U/ml). After 24 h incubation, BDNF, NT3 and NT4 levels were measured by means of ELISA methods(Promega Inc, USA). Both cell alone group and LPS group were used to consist of control groups to compare with the others. In BDNF and NT3 levels, there were no significant difference between the control group and the other groups. LPS+IFN , AMY , microglial activation by LPS+IFN+EPO and EPO treatment enhanced the NT4 levels compared to the basal levels. That AMY enhanced the NT4 expression in microglial cells made us think about this increase might be a reactive response against to the damage of toxic agent. On the other hand, the effect of EPO treatment might be probable by augmenting neurotrophic factor expression in microglia cells which supports neuronal survival.

Filiz KORKMAZ¹, Feride SEVERCAN²

Interactions of progesterone with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) were investigated as a function of temperature and progesterone concentration by using three non-invasive techniques namely Fourier transform infrared (FTIR) spectroscopy, turbidity at 440 nm and differential scanning calorimetry (DSC). DSC and turbidity studies and the investigation of the C-H, C=O and antisymmetric double stretching modes in FTIR spectra reveal that progesterone changes the physical properties of the DPPC bilayers by decreasing the main phase transition temperature, abolishing the pretransition, disordering the system in both gel and liquid crystal phase, increasing the dynamics of the system for low concentrations whereas stabilizing the acyl chains for high concentrations and inducing phase separation for low concentrations. Progesterone does not cause any hydration in C=O groups, whilst it makes significant hydrogen bond between groups and the water molecules around. Results lead to the conclusion that progesterone intercalates into the hydrophobic core of the membrane.

PURIFICATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM SIX-MONTH-OLD LAMB KİDNEY CORTEX

Ulusu N.N., Tandoğan B.

Hacettepe University, Faculty of Medicine, Department of Biochemistry, 06100 Ankara/Turkey

Glucose-6-phosphate dehydrogenase (D-Glucose-6-phosphate: NADP⁺ oxidoreductase EC 1.1.1.49) catalyses the first and rate limiting step in the pentose phosphate pathway. We purified glucose-6-phosphate dehydrogenase from six-month-old lamb kidney cortex for the first time. By the other authors, a variety of methods consisting of numerous steps have been applied to obtain a reasonable amount of pure enzyme from other organisms and tissues. In this study the purification procedure composed of two steps after ultrasantrifugation. We used 2’, 5’-ADP Sepharose 4B affinity and DEAE Sepharose Fast Flow anion exchange chromatography for rapid and easy purification. Previously, we used this procedure for the purification of glucose-6-phosphate dehydrogenase from bovine lens. One problem was to overcome the separation of glucose-6-phosphate dehydrogenase from 6-phosphogluconate dehydrogenase since the latter enzyme could be bound to 2’, 5’-ADP-Sepharose 4B column. But, this time 6-phosphogluconate dehydrogenase was not bound to the affinity column. The enzyme was purified from lamb kidney cortex, about 3640 fold with an overall yield 26.32 %. The enzyme was stable at 4ºC for a week.

Key words: Glucose-6-phosphate dehydrogenase, lamb kidney cortex and purification

P309

IS INSULIN LIKE GROWTH FACTOR-1 RELATED WITH THYROID FUNCTION TESTS ?

Alev USLU, Ayşenur ATAY, Banu ARSLAN, Erkan SOGUT , Füsun ERCIYAS

Recent experimental studies have shown that there may be some differential effects of IGF-1 like as IGF-1 induced mitogenic effects on thyroid epitelial cell. Insülin, IGF-1 and TSH receptors have been linked to synergistic cascade response system of the thyroid involving growth, thyroglobulin biosynthesis and thyroid hormone formation. The aim of the present study was to investigate the relatioship between serum IGF-1 and thyroid hormone levels in euthyroid, hypothyroid and hyperthyroid patients.

Thirty patients were divided into three groups according to their TSH levels and clinical manifestations ; Group I- Hyperthyroid patients, TSH<0.35 uIU/ml (n=11); Group II- Euthyroid patients, TSH= 0.35-5.5 uIU/ml (n=10) ; Group III- Hypothroid patients, TSH> 5.5 uIU/ml (n=9) . Serum thyroid function tests were determined with electrochemiluminesans assay on ACS Centaur autoanalyser and IGF-1 levels were measured by non extraction IRMA.

SPSS (Version 6.0) for Windows was used for istatistical analysis with Kruskall-Wallis, One way ANOVA and Mann Whitney U tests.

A strong inverse relationship between age and IGF-1 levels (r= 0.6767, p=0.000) and T3 and IGF-1 (r=0.4965, p= 0.031) by Pearson correlation. We found the negative correlation between TSH and IGF-1(r=-0.343, p=0.05) in thirty patients. There weren’t any correlations between IGF-1 and gender, T4, FT3, FT4. IGF-1 levels were significantly higher in Group I (668.25+/-278 ng/ml, p<0.009 ) than Group III (386.35+/-142 ng/ml). IGF-1 levels were significantly higher in Group II (619.35+/-222 ng/ml, p<0.026) than Group III. There was a significant difference (p= 0.0194 )among three groups for IGF-1 levels.

These results suggest that IGF-1 levels are affected by thyroid dysfunction. We demonstrated serum IGF-1 levels were positively related with T3 and negatively related with TSH. IGF-1 may be a naturel TSH inhibitor and T3 may stimulate the hepatic production of IGF1. In future, more research is needed to determine the mechanism of these hormone effects.

Mehmet AKÖZ, Mehmet GÜRBİLEK, Volkan KOCABAŞ, Cemile TOPÇU

When making comment on the laboratory results the values accepted as normal value is very valuable. İndepented laboratories wish to obtain and use their own referance ranges.

This values and their limits cover 95% of the population and accepted and evaluated within the mean values and standart deviations. So many parameters may effect and change the levels and limits; for example age, sex, race, enviroment, daily habits and siclus changes, food and drugs intake and excercises. In this study, we study to determine the reference ranges of CEA, Ca 15-3, Ca 19-9, Ca 125, Ca 72-4, AFP tests performed in our laboratory.

Patients admitted to our hospital for diagnostik purposes were divided in to subgroups regarding to their age, sex and other faetures. Blood samples were obtained early morning with vacutainer tup. The serum and plasma were seperated as soon as possible. The reliability of the results were checked by internal and external control programs.

The Analysis were performed by Chemiluminisans Immunassay method using Immulite one and ımmulıte 2000 hormon analyser and DPC hormon kits.

If the analysis results are over SD₃ the cases eliminated from the study. CEA for man: n=2250, =2.63, M=2.0, SS=1.88, S₁₌0.12-3.88, for woman: n=2250,=1.98, M=1.4, SS=1.63, S₁=0,01-3,03. for Ca 15-3; n=2700,=27.62, M=27.4, SS=10.67, S₁₌16,73-38,07. for Ca 19-9; n= 2700, =11.39, M=9.4, SS= 7.14, S₁=2,26-16,54. for Ca 125; n=2700, =8.99, M=8.3, SS=3.93, S₁=4,37-12,23. for Ca 72-4; n= 1210, = 2.24, M=1.74, SS= 1.85, S₁₌0,01-3,59.for AFP; n= 3150, = 1.92, M=1.51, SS= 1.37, S₁₌0,14-2,88.

The reference ranges of the tumor marker tests are determined in Konya region.

P311

THE EFFECT OF SULFITE OXIDASE DEFICIENCY ON RAT HIPPOCAMPUS ANTIOXIDANT STATUS

Vural Kucukatay¹, Feyza Savcıoğlu¹, Gülay Hacıoğlu¹, Melek Bor-Küçükatay¹, Piraye Yargıçoğlu², Aysel AĞAR<sup>1

1</sup>Akdeniz University, Faculty of Medicine, Department of Physiology

²Akdeniz University, Faculty of Medicine, Department of Biophysics

Sulfites are added to foods for a variety of important technical purposes, including the control of enzymatic and non-enzymatic browning, antimicrobial actions. Considerable quantities of sulfite are also generated in the body by normal catabolic processing of sulfur-containing-amino acids and other sulfur-containing compounds. Regardless of the source, Sulfite is a toxic molecule and can react with a variety of humoral and cellular component and can cause toxicity. Little information is available about the mechanism of sulfite toxicity in the body, but its damaging effect to the cellular components may involve formation of sulfur- and oxygen centered free radicals. For this reason it is oxidized to sulfate ion, a reaction catalysed by the enzyme sulfite oxidase. There are significant differences among species in their sulfite oxidase activity. Most notable is the difference between rat and man, with the latter reported to possess only about 5-10 % of hepatic activity of the rat. Although the rats have been used predominantly in the past for evaluation of sulfite toxicity, this species may not be the most appropriate model available for the prediction of sulfite toxicity in man. Rat tissues can be depleted of sulfite oxidase activity by maintaining animals on a regimen high in tungsten and low in molybdenum. It has been suggested that these rats might be used as a model for the prediction of sulfite toxicity in human.

The main objective of this investigation was to study effect of sulfite oxidase deficiency protocol on rat hippocampus antioxidant status. This study was conducted in normal and sulfite oxidase deficient rats. The rats were made deficient in sulfite oxidase by the administration of a high-tungsten/low molybdenum regimen for a period 21 days. At the end of this period, both groups of rats were killed by exsanguinations under urethane anesthesia. Their livers and hippocampus were removed for assessing sulfite oxidase, antioxidant enzymes (SOD, CAT and GPx) and Thiobarbituric acid Reactive Substance (TBARs) levels.

Deficency protocol was very effective in reducing sulfite oxidase activity. Sulfite oxidase activity in rats treated with high-tungsten/low molybdenum regimen decreased about approximately level of 1 % of the control group on 21^th. No significant antioxidant enzymes (SOD, CAT GPx) activities and Thiobarbituric acid Reactive Substance (TBARs) levels were seen in both of groups. These parameters were not changed by deficiency protocols. In summary, we proposed that sulfite oxidase deficiency has no effect on rat hippocampus antioxidant status.

Piraye Yargıçoğlu¹ , Selçan Aydın¹, , Narin Derin¹, YakupAlıcıgüzel³, Osman Elmas.³ Aysel Ağar²

There is accumulating evidence to indicate that stress can stimulate numerous pathways leading to an increased production of free radicals. The other factor that leads to lipid peroxidation, is sulfite compounds that are widely used as preservatives in foods, beverages and pharmaceuticals. The effects of sulfite and stress together on the lipid peroxidation and antioxidant status have not been previously studied. Therefore, the present study was undertaken to investigate the effects of stress and/or sulfite on lipid peroxidation and antioxidant status. Forty male albino rats, aged three months, were equally divided into four groups :Control (C), the group exposed to restraint stress (R), the group treated with sulfite (S) and the group exposed to stress and treated with sulfite (RS). Rats were exposed to 1 hour of restraint stress daily for 21 days by placing the animals in a 25x7 cm plastic bottle .Sodium metabisulfite (520 mg/kg/day) was given by gavage to the S and RS groups for 21 days. After the end of the experimental period, Cu, Zn-superoxide dismutase (Cu, Zn-SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and thiobarbituric acid-reactive substances (TBARS) levels of brain were measured. TBARS levels were significantly increased in all experimental groups with respect to the C group, but higher in the RS group than in the R and the S groups. Cu, Zn-SOD activity was found to be decreased in the R group, but increased in the S group.compared with the C group. However, in the RS group it was unaltered. Brain GSH-Px activity was significantly decreased in the R and S groups with respect to the C group. Statistically significant decrement in the brain Cu, Zn-SOD level was not detected in the RS group compared with the C group. Brain CAT activity was observed to be lower in the R and HS groups than the C and S groups. Our results show that stress and sulfite resulted in an increase in the lipid peroxidation process, that is accompanied by changes of antioxidant enzymes.

İpek ŞAHİN¹, Nadide Kazancı¹ and Feride Severcan²

Melatonin is an lipophilic antioxidant drug which is widely used for the prevention from several diseases. In the present study we will report the results of melatonin induced changes occuring in dipalmitoyl phosphatidylcholine (DPPC) membranes using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) .

Infrared spectra were obtained using a Bomem 157 FTIR Spectrometer which was continiously purged with dry air. The spectra were recorded in the 4000-1000 cm^-1 region with CaF₂ window using 12 m path length. Interferograms were accumulated for 50 scans at 2 cm^-1 resolution. The Grace-Specac temperature controller unit was used for temperature regulation. Bomem Easy software was used for all FTIR data manipulations. For DSC studies, a TA Q100 DSC instrument was used with a heating rate of 1^C/min.

The infrared spectra of DPPC multilamellar liposomes, both pure and containing different concentration of melatonin were investigated as a function of temperature. The C-H stretching, the C=O stretching and PO^-₂ antisymmetric stretching mode were considered.

The results of both FTIR and DSC studies reveal that melatonin changes the physical properties of the DPPC bilayers by decreasing the main phase transition temperature, abolishing the pretransition, ordering the system in the gel phase, increasing the dynamics of the system and causing strong hydrogen bonding in between the C=O and P=O groups of DPPC and either melatonin or the water molecules, both in the gel and liguid crystalline phases. Furthermore melatonin, at high concentrations, induced phase separation in DPPC membranes.

This work has been partially supported by Ege University Research Fund AFP: 2002 Fen 025

Gargılı, A¹, Kandil, A.², Çetinkaya, H³, Atukeren, P⁴., Demirci, C²., Gümüştaş, M.K⁴.

Toxocara canis is a nematod found in dog intestine. Paratenic hosts including human beings can be infected by the ingestion of embrionated eggs of the nematode shed with the dog faeces, via contaminated food or water. Larvae migrate in various organs such as liver, lungs, brain and eyes. During larval toxocariosis, migrating larvae cause pathological disorders which are known as Visceral Larva Migrans (VLM) in humans and other paratenic hosts.

Filiz Kuralay¹, Coşkun Yildiz, Omer Ozutemiz, Huray Islekel¹, Sezer Caliskan¹, Basak Bingol¹, Sermin Ozkal²