13th balkan biochemical biophysical days & meeting on metabolic disorders’ programme & abstracts

CONJUGATION Semiquinone Quinones

Binding of macromolecules DNA, RNA and proteins

Cellular necrosis Mutagenesis Malignant transformation

In this study, 3 months Adult male New Zealand white rabbits (1.5-2 kg) were injected with a single dose of alloxan (100 mg/kg) to induce diabetes. Six weeks later, diabetic and control rabbits were killed by decapitation and liver microsomes were prepared by differential centrifigation and stored in liquid nitrogen tank. The hydroxylation of p-nitrophenol to 4-nitrocatechol was determined in an assay medium containing 100mM Tris-HCl buffer, pH 6.8, 0.25 mM p-nitrophenol, 1.5 mg microsomal protein and 0.5 mM NADPH generating system in a final volume of 1.0 ml. The reaction was carried out at 37 ^oC for a period of 10 minutes. Western-blot analysis of control and diabetic rabbit liver microsomes were carried out using anti-rabbit CYP2E1 antibodies. Student’s t-test was used for statistical interpretations of the results, and P<0.05 was chosen as the level of significance.

The fasting blood glucose levels of control and diabetic animals was found to be 140 ± 0.58 mg /dl (mean ± SEM, n=3) and 506 ± 56 mg/dl (n=10), respectively. The results showed that the intravenous injection of single dose of alloxan induced the diabetus mellitus as determined by about 4-fold increase in the blood glucose level. Induction of diabetes caused a significant 1.3-fold increase in the cytochrome P450 content of liver microsomes. Induction of specific CYP2E1 was demonstrated by western-blot analysis and a single protein cross reactive with anti-rabbit P4502E1 antibody was observed in diabetic and control liver microsomes. The intensity of this protein band was markedly increased in the diabetic rabbit liver microsomes compared to control. An average p-nitrophenol hydroxylase activities were found to be 0.63 ± 0.05 nmole product/min/mg protein (n=10) for liver microsomes obtained from the diabetic rabbits and 0.35 ± 0.026 nmole product/min/mg protein (n=3) for control rabbits. These results showed that, diabetes caused significant 1.8-fold increase in p-nitrophenol hydroxylation rates of rabbit liver microsomes. Therefore, p-nitrophenol hydroxylase induction in diabetes may cause stimulation of semiquinone and quinone formation in turn this may increase the risk of hepatocellular damage, mutagenesis and malignant transformation.

P228

POLYMORPHISMS OF HUMAN DRUG METABOLIZING ENZYMES: STUDY OF DraI GENETIC POLYMORPHISM OF CYP2E1

Gülen ULUSOY, Emel ARINÇ, Orhan ADALI

CYP2E1, an isozyme of P450 superfamily, is induced by ethanol, benzene and pyridine and catalyzes chlorzoxazone-6-hydroxylation reaction. It also takes role in the metabolism of N-nitrosamines, benzene, urethane, acetone, halogen hydrocarbons and other low molecular weight compounds. It has been shown that CYP2E1 increases the cancer risk as it converts procarcinogens into carcinogenic forms. This enzyme shows polymorphisms which are thought to be associated with incidence of cancer risk. One type of CYP2E1 polymorphism is the SNP at position 7632T>A in intron 6, which can be detected by RPLF using DraI restriction enzyme. Studies have shown an association between DraI polymorphism and incidence of lung, breast cancer and renal carcinoma.

In this study, DNA was isolated from blood samples belonging to healthy Turkish individuals by phenol:chloroform:isoamylalcohol extraction method. Qualitative analysis of DNA was performed by 0.4% agarose gel electrophoresis and the concentration and purity of the samples were determined spectrophotometrically by measuring the absorbance values at 260 and 280 nm. To amplify the DraI restriction site on intron 6 of CYP2E1 gene, the optimized PCR medium contained 10mM Tris-HCl, 50 mM KCl, 0.08% Nonidet P40, 1.5 mM MgCl₂, 0.2 mM dNTP mixture, 20 pmol of each primer (5’-TCGTCAGTTCCTGAAAGCAGG-3’ and 5’-GAGCTCTGATGCAAGTATCGCA-3’), 0.4 U of Taq DNA polymerase and 200 ng of DNA. The cycles of PCR were optimized as follows: 94C, 3 min. for denaturation; 61C, 1 min for annealing; 72C, 1 min for extension and a final extension at 72C for 6 min. The PCR product of nearly 1000 base pairs was detected by 1.5% agarose gel electrophoresis. For the detection of polymorphism, RFLP was performed on the PCR products. 20 µL of PCR product was incubated with 4 U of restriction enzyme DraI at 37C for 18 hours and the results were visualized by 1.8% agarose gel electrophoresis.

Results showed that amplified region on intron 6 contained one DraI restriction enzyme site in wild type. After DraI digestion, and gel electrophoresis; the individuals were classified as DD for homozygous wild type, CC for homozygous mutant type and CD for heterozygotes according to the band patterns on the gel. Homozygous wild type, homozygous mutant type and heterozygote individuals gave two bands of 600 bp and 300 bp, a band of 900 bp and three bands of 900, 600 and 300 bp upon digestion with DraI, respectively. In all cases, PCR products gave a minor 100 bp band when digested with DraI.

Among the 200 blood samples collected, DraI RFLP analysis for 36 have been completed. 31 of the subjects had DD genotype, 5 of them had CD genotype and none had CC genotype. The wild type allele frequencey was found as 93.1%, whereas the mutant allele (CYP2E1*6) frequency was 6.9%.

P229

BIOCHEMICAL MONITORING OF TOXIC AND CARCINOGENIC ORGANIC POLLUTANTS ALONG THE İZMİR BAY AFTER THE GREAT CANAL PROJECT AND POSSIBLE HEALTH EFFECTS

Tuğba BOYUNEĞMEZ, Azra BOZCARMUTLU, Emel ARINÇ

Graduate Program in Biochemistry, Department of Biology, Middle East Technical University, 06531, Ankara, TURKEY

btugba@metu.edu.tr

The İzmir Bay is located on the Aegean sea of the Mediterranean Sea. Industrial wastes, urban and agricultural run off, discharges from ships, and waters of rivers have contaminated the Bay by the heavy metals and the organic chemicals including PAHs, PCBs, dioxins and pesticides. Amoung these pollutants, PAHs, PCBs, dioxins induce one family of P450, P4501A in fish liver. P4501A oxidatively metabolize these toxic compounds, precarcinogen/ carcinogens to their epoxides and other oxygenated metabolites which in turn bind to DNA and form DNA adducts leading to membrane impairment, cellular toxicity, mutation or even carcinogenesis. The induction of hepatic CYP1A and its monooxygenase activity 7-ethoxyresorufin O- deethylase (EROD) in fish by PAHs, PCBs and dioxins has been suggested as an early warning system, a “most sensitive biochemical response” for assessing environmental contamination conditions. This has implications for human fish consumption, as well as for the health status of the organisms.

This study was carried out to determine if there exists a decrease in the concentrations of PAHs, PCB and dioxins after the Great Canal Project in the İzmir Bay by measuring induction of cytochrome P4501A associated EROD activity and to compare these results with the previous studies. (Arınç and Şen, Marine Environmental Research.; 48, 147, 1999, Arınç, Şen and Bozcaarmutlu, Pure Appl.Chem.; 72, 985, 2000.).In this study, two types of fish species, Leaping Mullet (Liza saliens) as a pelagic fish and Annular sea bream (Diplodus annularis) as a benthopelagic fish were examined for cytochrome P4501A associated 7- ethoxyresorofin O-deethylase (EROD) activity. Fish were captured on November 2002 from different sites of the Bay. Microsomes were prepared from liver by differential centrifugation. EROD activity of microsomes were determined spectrofluorometrically. Mullet caught from outer site of Bay had very low EROD activity (34 pmol/min/mg, n=2). Fishes from the Inner Bay (Liman) which has a long term history as sinks for petroleum hydrocarbons, showed elevated EROD activity (2258840, pmol/min/mg, n=15); about 66 times higher with respect to the value of outer site of the Bay. Mullet caught along a pollutant gradient at two other region: Pasaport (Inner Bay) and Üçkuyular (Middle Bay) also displayed highly elevated EROD activities which were 1813951 (n=11) and 14001039 (n=7) respectively and about 53 and 41 times higher than those obtained from the outer site of the Bay. EROD activities of Annular sea bream that caught from the Outer Bay were low (14664 pmol/min/mg, n=7), while EROD activities of fish captured from Pasaport (Inner Bay) and Üçkuyular (Middle Bay) were highly elevated (758336 n=8 , 706372 n=8 pmol/min/mg respectively).

Although, İzmir Great Canal Project has been going on to treat and protect the İzmir Bay from the contamination of domestic and industrial wastes since 2000, highly elevated liver EROD activities of mullet and seabream in the Inner and Middle Bay of İzmir indicated that concentrations of PAHs and PCB type toxic and carcinogenic pollutants are as high as before the Great Canal Project. Human consumption of fish from Inner and Middle Bay may result toxic effects and even carcinogenecity in humans.

THE EFFECTS OF ALCOHOL AND SMOKING ON SERUM, SALIVA, AND URINE SIALIC ACID LEVELS

Naciye Kurtul^a, Aynur Arıcan^a, Ebubekir Bakan<sup>b

a</sup>Department of Chemistry, Faculty of Science, KSU, Kahramanmaraş/TURKEY

^bDepartment of Biochemistry, Medical School, Atatürk University, Erzurum/TURKEY