ANTIOXIDANT EFFECT OF GREEN TEA AND COENZYME Q10 IN HUMAN CORONARY ARTERY ENDOTHELIAL CELLS Pureda Yazici1,2, Oya Sayin1, Gul Guner1,2,3 Dokuz Eylul University School of Medicine Research Laboratory (ARLAB)1, Dokuz Eylul University Health Sciences Institude2, Dokuz Eylul University School of Medicine Biochemistry Department3 Izmir/TURKEY drpuredayazici@yahoo.com Introduction: There is 100-200 mg EGCG (epigallocatechin 3-gallate in a cup of green tea. EGCG is the most abundant component of green tea nearly about %65 and most potent one because of its galloyl group. Polyfenon 60 (P60); is the combination of green tea components like epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin 3-gallate (EGCG) (1). Green tea and coenzyme Q10 (CoQ10) are known by their antioxidant properties. In this study our aim is to investigate the effect of EGCG, P60 and CoQ10 to the in vitro Hypoxia-reoxygenation induced coronary artery cell injury. Materials and Methods: Human Coronary Artery Endothelial Cells (HCAEC) are supplied from Cambrex and Promocell. Cells are growth in Endothelial Growth Medium (EGM-2). Hypoxia-reoxygenation (H-R) is applied in different time protokols and it is tried to find the time that cause to the ROS induction. It’s known that after a time of hypoxia, reoxygenation cause to ROS formation from O2. Hypoxic chamber is aerated with 95% N2, 5% CO2 and incubated at 37oC for forming 1% O2 saturation as hypoxic condition. For reoxygenation the cells incubated in the 95% air 5% CO2 incubator at 37oC. In hypoxic period PBS is used for also forming glucose deprivation. In incubation with EGCG, P60 and CoQ10 and in reoxygenation period EGM-2 medium is used. Results: 6h hypoxia and 1-4-18h reoxygenation didn’t cause to ROS induction. 2h incubation before 6h hypoxia and 1-4-18h reoxygenation with 50- 100-200 µM EGCG, 31.25, 62.5, 125 µg/ml P60, 10-50 µM CoQ10 didn’t cause to significantly important change on ROS levels. Data not shown. When (24h hypoxia 15min-1h-3h) reoxygenation applied, in (24h hypoxia 15min and 3h reoxygenation) cause to increase on ROS levels significantly (p=0.029, p=0.029). (24h hypoxia and 1h reoxygenation) didn’t change ROS levels (Fig 1). 24h hypoxia+ 15 min) reoxygenation is increased ROS according to the (24h + 15 min) normoxia (p<0.029). [24h incubation with (100-200- 500-2000µM) EGCG +24h hypoxia + incubation] is applied. It is seen that at any µM, EGCG didn’t alter ROS levels(p=0.629,p=0.857, p=0.0229, p=1.000) (Fig 2). [24h incubation with (50-100-500 µg/ml) P60 + 24h hypoxia + incubation] is applied. 50-100-500 µg/ml P60 didn’t change ROS levels significantly. 1000 µg/ml P60 was toxic to the cells and induced the ROS levels (p=0.034) (Fig 3). [24 h incubation with (5-10-50-100 µM) CoQ10 +24h hypoxia + incubation] is applied. 5 µM CoQ10 decreased the ROS levels significantly (p=0.034). 10-50-500 µM CoQ10 didn’t change ROS levels p=0.267, p=0.857, p=0.400) (Fig 4). Discussion: In our hypoxia-reoxygenation model 24h hypoxia and 15min and 3h reoxygenation increased ROS levels but not 1h reoxygenation. EGCG at any dose (100-200-500-2000 µM) didn’t alter ROS levels. P60 at high dose(1000 µg/ml) increased ROS levels (p=0.034) 5 µM CoQ10 decreased ROS levels p=(0.034). It is shown that potassium superoxide and alkaline autooxidation of green tea cause to genetation of free radicals (2). Halliwell B reviewed that EGCG can be also pro-oxidant in culture media and cause production of H2O2 because of autooxidation (3). 1-10 µM coenzyme Q10 decreased ROS levels in HUVEC (4). References: Zaveri NT. Green tea and its polyphenolic catechins: Medicinal uses in cancer and noncancer applications. Life Sciences 78(2006):2073-80. Severino JF, Goodman BA, Kay CWM, Stolze K, Tunega D, Reichenauer, Pirker KF. Free radicals generated during oxidation of green tea polyphenols: Electron paramagnetic resonance spectroscopy combined with density functional theory calculations. Free Radical Biology & Medicine xxx (2008) xxx-xxx. Halliwell B.Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? Archives of Biochemistry and Biophysics? 476;(2008):107-12. TsunekiH, Sekizaki N, Suzuki T, Kobayashi S, Wada T, Okamoto T, Kimura I, Sasaoka T. Coenzyme Q10 prevents high glucose-induced oxidative stress in human umblical vein endothelial cells. European Journal of Pharmacology 566:(2007):1-10. Figure 2. [24h incubation with EGCG+ 24hypoxia (H)+incubation with EGCG+ 15 min reoxygenation (R)] N: normoxia Figure 1. [24h hypoxia (H) + 15 min -1h-3h reoxygenation (R)] N:normoxia Figure 3. [24h incubation with P60+ 24 hypoxia (H) + incubation with P60+ 15 min reoxygenation (R)] N: normoksia Figure 4.[24h incubation with CoQ10+ 24 hypoxia (H)+incubation with CoQ10+ 15 min reoxygenation (R)] N: normoxia This project is supported by DEU BAP (Scientific Research Projects) (Project Number:2006. KB. SAG.009