Mol Biotechnol (2007) 37:81–97 DOI 10.1007/s12033-007-0058-5

ABSTRACTS

Abstracts

Published online: 7 August 2007
Humana Press Inc. 2007

Platform Presentation Free Radicals and Blood Rheology Yousif Y. Bilto Department of Biology & Biomedical Sciences, Jordan University, Amman, Jordan In vivo circulating erythrocytes and leukocytes are constantly exposed to both intracellular and extracellular sources of free radicals. When production of free radicals overwhelm the capacity of antioxidant systems in the erythrocytes, oxidative damages may occurs endangering the integrity of the erythrocytes. Oxygen radical generating systems such H2O2 and ascorbate/iron have been used as sources of oxygen radicals in in vitro studies. In vitro exposure of erythrocytes or neutrophils to oxygen radical generating systems was shown to induce lipid peroxidation, protein degradation, loss of deformability and an increase in osmotic fragility and oxidant hemolysis of erythrocytes. Free radicals and lipid peroxidation have been implicated in over 100 human diseases, but the in vivo hemorheological implications associated with increased production of free radicals have not been investigated in depth. The lecture will focus on the results of the ongoing research in our laboratory which concerned with the effects of free radicals on erythrocyte rheology, neutrophil rheology, lipid peroxidation, protein degradation and neutrophil chemotaxis and random migration. Also, the susceptibility of erythrocytes from NIDDM, hemodialysis patients, cigarette smokers to oxidant stress and loss of rheology were studied. The results demonstrate a direct relationship between increasing oxidant stress

and loss of erythrocyte and neutrophil rheologies and also showed clearly that the oxidant-stress-induced loss of erythrocyte and neutrophil rheologies were caused mainly by protein degradation rather than by lipid peroxidation. Neutrophils exposed to free radicals also showed reduction in chemotaxis and random migration. As plants could represent a source of natural compounds with antioxidant activity, we were able to screen selected medicinal plants and flavonoids for their protective activity against protein degradation, lipid peroxidation and deformability loss of human erythrocytes exposed to H2O2. The results of these studies showed that the antioxidant activity of some selected medicinal plants varied from being either anti-lipid peroxidant, anti-protein degradant or having both activities, probably reflecting the structural variation of the antioxidant compounds involved. The studies on selected flavonoids clearly indicated the importance of the chemical structure in dictating the type of antioxidant activity and also indicated the hemorheological potentials of natural compounds that have particular protein-antioxidant activities. The overall results demonstrate the importance of oxidatively damaged cellular proteins in compromising the rheologic behavior of blood cells, particularly under oxidative stress conditions.

Modeling Copper Trafficking Proteins and New Perspective on Oxidative Stress Sabyasachi Sarkar Department of Chemistry, Indian Institute of Technology Kanpur, India, Fax: +91-512-2597265; E-mail; [email protected]

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The advancement of human civilization parallels the use of metallic copper since the Neolithic times. Copper is an imperative metal in life; in contradiction, however, it is highly toxic. Copper ions play crucial roles for the sustenance of aerobic life as of dioxygen respiration in cytochrome c oxidase and in detoxifying super oxide dismutase. Aerial oxygen utilization mediated by copper(I) in the Wacker process is also known Nature uses copper ions in several other forms of oxidases and also in transporting dioxygen by hemocyanin in varied domains of life. Notable among these is the reaction of Cu (I) with dioxygen which spurred chemists directing toward elucidating the structure-reactivity through synthetic model systems. Such reactivity involving O–O bond cleavage is ubiquitous among most N-coordinated Cu (I) complexes. Dioxygen binds to dicopper(I) centers as end-on, side-on forming dicopper(II) units or through the bridging mode even with the formation of a short lived dicopper(III) species under low temperature. A mismatch in copper ion and dioxygen chemistry in humans develops oxidative stress by generating reactive oxygen species (ROS) like O2 . This promotes the onset of ageing accompanied with serious diseases. In the present talk synthetic modeling of copper-chaperones which distribute copper to specific intracellular destinations will be discussed first. Then the spontaneous generation of superoxide ion in the metallation of a few bio relevant macromolecules like carrole and porphyrinogen with {N4} chelation by copper ion will be highlighted. This is in contradiction to the established chemistry of copper (II) in dismuting superoxide radical ion. This antagonism with {CuII(N4)} core containing species will be discussed to account for overall oxidative stress beyond SOD. Funding of this research by the Department of Science and Technology, Government of India, New Delhi is gratefully acknowledged. The Role of Green Tea in Protecting the Intestinal Mucosa of Fasting Animals from Free Radical-induced Damage Asfar Sami, Abdeen Suad, Mathew Thazhumpal, Dashti Hussein Faculty of Medicine, Kuwait University, Kuwait Objective: Fasting is known to cause intestinal mucosal damage which could breach the intestinal barrier and contribute to migration of microorganisms. In a previous histological study we showed that drinking green tea for 2 weeks prior to fasting, protected the intestinal

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mucosa from fasting-induced damage. The objective is to study the mechanisms involved in the protective effect of green tea on the intestinal mucosa. Methods: Four groups of male Wister rats were used (n = 12 per group): G1: Normal controls, on rat chow diet and water ad libitum. G2: Animals on rat chow diet and water ad libitum were fasting for 3 days (only i.p. 10% glucose 40 ml/day). G3: 2 weeks of drinking green tea solution ad libitum (instead of water) & rat chow then fasted for 3 days. G4: 2 weeks of drinking Vit. E-containing solution ad libitum (instead of water) & rat chow then fasted for 3 days.

On day 4 of fasting, blood was collected for biochemical analysis of total plasma antioxidants following which the animals were euthanized and 2 inches of jejunum was removed for analysis of SOD and GPx by immunohistochemical methods and MPO by biochemical analysis. Results: Compared to G2 (fasting) group, G3 rats showed: (1) an increase (P < 0.001) in total plasma anti-oxidants; (2) an increase (P < 0.001) in SOD, GPx and a decrease (P < 0.001) in the level of MPO in the intestinal mucosa. Although Vit. E showed an increase the level of total plasma anti-oxidants; it was not effective in inducing changes in the level of SOD, GPx, and MPO. Conclusion: Pretreatment with green tea protects the intestinal mucosa of fasting rats from free radicalsinduced damage. Correspondance to: Sami Asfar, M.B.,Ch.B., M.D.(UK), FRCSEd, FRCS, FACS, Professor & Chairman, Department of Surgery, Faculty of Medicine, Kuwait University, P.O.Box 24923 Safat-13110, Kuwait, Fax: (965) 531 9597

The Effect of Lack of Glycaemic Control in Diabetes Mellitus on the Ontogeny of Human Sperm is Through Oxidative Stress Alexander E Omu 1, Elijah O. Kehinde 2, Magedah K Al-Azemi 1, Florence E Omu 3, Chacko Mathew 4, Jehoram T Anim 5, Tunde Fatinikun 1 Departments of Obstetrics and Gynaecology1, and Surgery2, Anatomy4 and Pathology5, Faculty of Medicine, Health Sciences Center, Kuwait University, and College of Nursing, PAAET3, Kuwait Introduction: Although Diabetes Mellitus has been associated with impairment of fertility in men, but the mechanism has not been clearly elucidated.

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Objective of study: To investigate the effect of poor glycaemic control on sperm quality in men with insulin dependent diabetes mellitus and the role of antioxidants. Materials and Methods: Thirty-one men with poor glycaemic control insulin dependent diabetes form the subjects of this study. Twenty-five non-diabetic infertile men matched for age and duration of infertility formed the control group. The study protocol included initial and post-therapy clinical evaluation of all the patients, semen analysis, hormone profile, HbA1C, Malonedialdehyde (MDA), Butyrylcholinesterase (BuChE), lipid profile, Acridine orange denaturation of sperm and light and electron microscopy. The patients were administered Zinc and vitamins E and C for 3 months. Results: Diabetes mellitus was associated with significantly impaired sperm motility (asthenozoospermia) compared to control (64 versus 36%, P < 0.05), normal morphology (66 versus 52%, P < 0.05), higher HbA1C (9.6 versus 4.4%, P < 0.05) and MDA (2.4 versus 1.4 nmol/L, P < 0.01). There was an inverse relationship between glucose level and BuChE (r = 0.486, P < 0.05). Antioxidant therapy significantly decreased glucose level, 18–40% P < 0.05; HbA1c 9–29% P < 0.05; MDA level 33–41%, P < 0.01; and Sperm DNA Fragmentation index, 23–33%, P < 0.01) and Increase in BuChE 21–40%, P < 0.05 and TAC, 27–36 %, P < 0.05. Conclusion: Poor control of Diabetes mellitus was associated with impaired sperm quality through oxidative stress that could be prevented by antioxidant therapy. Key words: Diabetes mellitus, Oxidative stress, Sperm Quality, Antioxidant Correspondence: Dr. Alexander E. Omu, Department of Obstetrics and Gynaecology, Faculty of Medicine, HSC, Kuwait University, PO Box 24923 Safat, 13110 Kuwait. Tel.: 5319601, Fax: 5338906 and e-mail: [email protected] Mn-Complex-Based Superoxide Diamutase Mimic Ameliorates Oxidative Stress in Alloxan-induced Experimental Diabetes Mellitus Mohammed A El-Missiry, Azza I Othman, *Tarek A Salem, *Ibrahim H El-Sayed I Zoology Department, Faculty of Sciences, Mansoura University, Mansoura and *Molecular Biology Department, Genetic Engineering and Biotechnology Institute, Minoufiya University, Egypt Mn-complex has superoxide dismutase (SOD) mimetic activity ameliorated oxidative stress cause by alloxaninduced diabetes. We used a pharmacological approach to ameliorate reactive oxygen species destruction

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associated with diabetes by using a synthetic metallomethylaminopyridine-based SOD mimic, Mn-complex. Accordingly, the present study focused on investigating the potential antioxidant role of Mn-complex against free radical-mediated damage in the liver and brain tissues of alloxan-induced diabetes. Adult male rats were injected intraperitoneally with 120 mg kg–1 of alloxan to produce experimental oxidative stress characteristic of diabetes mellitus. Hyperglycaemia, hypercholesterolaemia, and hypertriglycerdaemia were observed in serum after 7 days of alloxan treatment. This is associated with a depression of glutathione concentration, superoxide dismutase, and catalase activities and increase glutathion-S-transferase activity in the liver and brain. In addition, the thiobarbituric acid-reactive substances are significantly elevated, indicating increased lipid peroxidation and oxidative stress in the same tissues. Alloxan-treated rats also showed increased levels of nitrite/nitrate in serum, liver, and brain as well as ascorbic acid in urine. The results revealed that oral administration of 10 mg kg–1 (body weight) of Mn-complex for 7 days had no effect on blood glucose, triglyceride and cholesterol levels but significantly increased the activity of superoxide dismutase and glutathion-S-transferase and markedly inhibited the increase in lipid peroxidation caused by alloxan in the liver and brain. In addition, significant reductions in the levels of nitrite/nitrate content in serum, liver, and brain total as well as urinary ascorbic acid levels were observed in alloxan-Mn-complextreated rats compared to the diabetic rats. Therefore, Mn-methylaminopyridine-based SOD mimic suppressed diabetes-induced oxidative stress, which presumably accounts for its potential beneficial effect on the health of the diabetic rats. The results indicate that SOD mimic can be used as a potent complementary therapeutic agent in diabetes.

Cadmium Exposure in Hypertensive Subjects Dr. Vinod George Thykadavil, *Dr. Justin V Gnanou *St. John’s Medical College, Bangalore, India, Fax: 918025502341, Phone: 918022065050, E-Mail: [email protected] An association between the chronic exposures to Cadmium (Cd) and hypertension (HT) has been established in this study. Elevated levels of urinary excretion of Cd in young hypertensive subjects without occupational exposure indicates a significant source of Cd from diet and environment in the population.

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Mol Biotechnol (2007) 37:81–97 Mean age of the study group: 29.73 ± 5.3 years Mean systolic and diastolic BP were 147.3 ± 11 94.5 ± 11.7 mmHg, respectively Urinary Cd content ranged from 4.2 to 30.36 lg/day

and

Cd was found to generate hydroxyl radicals, resulting in the damage to mitochondria and causes mitochondrial swelling, though Chronic Cd exposure and its role in the etiology of HT not fully elucidated ‘Oxidative stress’ has been proposed for the development of nephropathy. Oxidative Stress and Disturbed Antioxidant Defense in Diabetic Gestation: An Experimental Approach Salim M, Oriquat G and El-Bassiouni EA Faculty of Pharmacy and Medical Sciences, Al-Ahliyya Amman University Amman, Jordan The combined stress of pregnancy and diabetes could be an important risk factor for the increased incidence of malformations observed in the embryos of pregnant female diabetic rats. In the present study, diabetes was experimentally induced by streptozotocin (STZ), 1 week prior to conception, and the embryos were examined during organogenesis. The results clearly indicate a definite over-production of reactive oxygen species (ROS) and disturbed antioxidant defence, particularly in embryos with congenital anomalis. Oxidative stress is demonstrated by accumulation of thiobarbituric acid reactive substances (TBARS), determined as malondialdehyde (MDA), and depletion of reduced glutathione (GSH). The half-cell redox potential, calculated from the glutathione data, indicate that although embryo cells from the control and diabetic groups were of the same chronological age, they were at different stages of development. The activities of the glutathione-related enzymes were also perturbed. Increased oxidative stress in the embryos was associated with higher activities of glutathione peroxidases and glutathione -S-transferase and decreased activity of glutathione reductase. The changes in the activities of these enzymes were coupled with reduction in the low-molecular weight antioxidants, vitamins C and E as well as selenium, in the experimental diabetic gestation group, particularly in malformed embryos. It is of interest to note that oxidative stress was also coupled in this case with inhibited gene expression of cyclooxygenase-2 (COX-2) and decreased levels of prostaglandin E2 (PGE2). In contrast, the levels of PGF2a increased. Such increase seemed to parallel the

intensity of oxidative stress. This indicates that PGF2a may be produced by a route independent of the COX pathway. The synthesis of PGF2a in this case probably proceeds through the isoprostane pathway under the influence of oxidative stress and increased ROS production. Understanding the role played by oxidative stress in malformations produced in experimental diabetic gestation and its effects on other signaling molecules and growth factors may help identify the risk factors that lead to the appearance of such malformation. The final aim of our series of experiments in this respect is to identify and try to alleviate deviations from normal of such factors, in an effort to prevent or minimize the appearance of congenital anomalies. Effectiveness of Melatonin in Ameliorating Adriamycininduced Cardiotoxicity and Nephrotoxicity AzzaI Othman, Mohammed A El-Missiry and Maher A Amer Zoology Department, Faculty of Sciences, Mansoura University, Mansoura, Egypt, Email: [email protected] Clinical chemotherapy with the anticancer antibiotic adriamycin continues to be limited by its cumulative dose-related diverse cytotoxicity. It inhibits protein synthesis and provokes prooxidant effects. Melatonin has recently been shown to have high antioxidative properties. We investigated the ability of melatonin to neutralize the oxidative damage induced by a single dose (10 mg/kg, i.p.) of adriamycin preceded (5 days) and followed (5 days) by pharmacological dose (10 mg/kg, i.p.) of melatonin. After the administration of a single dose of adriamycin to male Wistar rats, glutathione, glutathione peroxidase, superoxide dismutase, catalase activities in the heart, and liver were significantly altered. This is associated with significant increase serum iron, transferrin, and ferritin. When the treatment of adriamycin was preceded and followed by melatonin, the changes in the antioxidants and serum iron, transferrin, and ferritin were significantly normalized. Significant increases in lipid peroxidation products and protein carbonyl formation were observed in heart and liver tissues after a single administration of adriamycin, which were attenuated by pre- and post-treatment with a melatonin. Our results demonstrate that oxidative damage induced by the antitumor drug, adriamycin, can be reduced by melatonin.

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Poster Presentation

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Effect of Superoxide on DNA

Effect of Green Tea Extract on Modulation of Reserpine Toxicity in Animal Model: Electron Microscopy and Bioanalytical Studies

Adel S. Wasfe

A. M. Safar, M. Afzal, and S. Al-Bloushi

Faculty of Pharmacy &Health Sciences, Ajman Universityof Science & Technology Network, Fujairah U.A.E, E-mail: [email protected] Superoxide promotes hydroxyl-radical formation and consequent DNA damage in cells of all types. The long-standing hypothesis that it primarily does so by delivering electrons to adventitious iron on DNA was refuted by recent studies in Escherichia coli. Alternative proposals have suggested that superoxide may accelerate oxidative DNA damage by leaching iron from storage proteins or enzymic [4Fe–4S] clusters. The released iron might then deposit on the surface of the DNA, where it could catalyze the formation of DNA oxidants using other electron donors. Whole-cell electron paramagnetic resonance demonstrated that the level of loose iron in superoxide-stressed cells greatly exceeds that of unstressed cells. Bacterial iron storage proteins were not the major source for free iron, since superoxide also increased iron levels in mutants lacking these iron storage proteins. However, overproduction of an enzyme containing a labile [4Fe–4S] cluster dramatically increased the free iron content of cells when they were growing in air. The rates of spontaneous mutagenesis and DNA damage from exogenous H2O2 increased commensurately. It is striking that both growth defects and DNA damage caused by superoxide ensue from its ability to damage a subset of iron-sulfur clusters.
Superoxide O2 , a by-product of aerobic metabolism, has been implicated in the production of oxidative DNA damage. Many redox-cycling drugs that
generate O2 are mutagenic to mammalian and bacterial cells. Furthermore, mutants of Escherichia coli that lack cytosolic superoxide dismutase (SOD) exhibit a high rate of aerobic mutagenesis, and SOD-deficient strains with attenuated DNA-repair systems cannot
grow in air at all. Although O2 is chemically incapable of damaging DNA directly, it is thought to do so indirectly by Participating in the production of hydroxyl
radicals (OHÆ). A long-standing proposal is that O2 serves as a reductant for iron that is adventitiously bound to DNA. Subsequent oxidation of the iron by hydrogen peroxide (H2O2) generates the hydroxyl radical, a powerful oxidant that attacks the adjacent DNA This mechanism has been confirmed to occur in model systems in vitro. It is also supported by the marked sensitivity of E. coli SOD mutants to killing by exogenous H2O2.

Department of Biological Sciences, Faculty of Science, Kuwait University, Kuwait This study investigated the possible role of green tea in restraining and reversing the damaging effect of antioxidant activity in rat hepatic cells and expediting cell revival caused due to oxidative stress. This experimental work was focused on some of the biochemical and histological aspects of green tea. Sprague–Dawely rats were intraperitonealy administered reserpine to induce oxidative hepatic damage. Experimental rats were given green tea extract according to the protocol given below. Sixty rats were randomly divided into six groups, with ten rats in each group. Reserpine was found to cause hepatic damage, with elevated level of oxidative stress markers, such as TBARS, transaminases and cholesterol. It also caused hepatic ultra-structural damage including cell membrane, cytoplasm, nucleus, nuclear envelope, rER, and mitochondria. Markers of oxidative stress and hepatic abnormal ultra-structural features were also found to be annulled by administering green tea extract. Biochemical results showed a decrease in TBARS, transaminases, and cholesterol level, while the histological study showed a revival of liver cells as a result of green tea extract administered to rats. The observed recovery could be due to free-radical scavenging activity of green tea. References 1. Abe, K., Ijiri, M., Suzuki, T., Taguchi, K., Koyama, Y., & Isemura, M. (2005). Green tea with a high catechin content suppresses inflammatory cytokine expression in the galactosamineinjured rat liver. Biomedical Research, 26, 187–192. 2. Afzal, M., Safer, A. M., & Al-Bloushi, S. (2005). CoQ9 potentiates green tea antioxidant activities in Wistar rats. BioFactors, 25, 255– 259. 3. Atoui, A., Mansouri, A., Boskou, G., & Kefalas, P. (2005). Tea and herbal infusions: Their antioxidant activity and phenolic profile. Food Chemistry, 89, 27–36. 4. Singal, A., Tirkey, N., Pilkhwal, S., & Chopra, K. (2006). Green tea (Camellia sinensis) extract ameliorates endotoxin induced sickness behavior and liver damage in rats. Phytotherapy Research, 20, 125–129.

Green Tea Polyphenols and Their Antioxidant Activities M. Afzal, A. M. Safar and S. Al-Bloushi Department of Biological Sciences, Faculty of Science, Kuwait University, Kuwait

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Polyphenols are ubiquitous in plants and so far over 2,000 plant polyphenolics, known as flavonoids, have been identified. These molecules are present either as glycosides or aglycones. Quercitin and rutin are the representative examples of these compounds. A variety of flavonoids have been found to inhibit tumor development in several experimental animal models and act through different mechanisms (1–4). In addition phenolic acids and tocopherols possess strong antioxidant activities that may contribute to their biological activities of these compounds (4). Tea is widely used as beverage by human and is prepared from leaves of Camellia sinensis. Tea leaves are used both as dried leaves called green tea, and also processed tea called black tea. Camellia sinensis is cultivated in many different parts of the world thus giving different composition to its extracts. Green tea extracts contain phenolic catechins, including flavonols (-)epigallocatechin gallate, (-)-catechin gallate, (-)-epigallocatechin, (-)-epicatechin and their glycosides. These flavonoids are present in large amounts in green tea and on daily basis, human can consume as much as 1 g of some of these compounds. Many studies have been reported about the antioxidant and antitumor activities of green tea aqueous extracts (5). We have undertaken to assess the total phenolic content of green tea leaves collected from different countries and have compared this with Korean red Gin Xing and other tea extracts. The antioxidant activities were assessed by DPPH and beta-carotene assay methods. It was found that green tea aqueous extracts were better antioxidant than any other tea extracts. Results of this comparative study on the antioxidant activities will be presented. References 1. Ohigashi, H., & Murakami, A. (2004). Cancer prevention with food factors: Alone and in combination, Biofactors, 22(1–4), 49– 55. 2. Oak, M. H., El Bedoui, J., & Schini-Kerth, V. B. (2005). Antiangiogenic properties of natural polyphenols from red wine and green tea. Journal of Nutritional Biochemistry, 16(1), 1–8. 3. Zhou, Y. D., Kim, Y. P., Li, X. C., Baerson, S. R., Agarwal, A. K., Hodges, T. W., Ferreira, D., & Nagle, D. G. (2004). HypoxiaInducible Factor-1 Activation by (-)-Epicatechin Gallate: Potential adverse effects of cancer chemoprevention with high-dose green tea extracts. Journal of Natural Products, 67(12), 2063–2069. 4. Ravindranath, M. H., Muthugounder, S., Presser, N., & Viswanathan, S. (2004). Anticancer therapeutic potential of soy isoflavone, genistein. Advances in Experimental Medicine Biology, 546, 121–165. 5. Huang, M.-T., & Ferraro, T. (1992). Phenolic compounds in food and cancer prevention. In M.-T. Huang, C.-T. Ho & C. Lee (Eds.), Phenolic compounds in food and their effects on health II, ACS symposium Series, 507 (pp 8–34). Washington, DC: American Chemical Society.

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Synergistic Effect of Green Tea Polyphenols with CoQ9 in Rats M. Afzal, A. M. Safar and S. Al-Bloushi Department of Biological Sciences, Faculty of Science, Kuwait University, Kuwait Camellia sinensis, a member of the Theaceae family is used both as green and black tea. Throughout the world, both forms of tea are used as a cold or hot beverage and since 3000 B.C., green tea has been consumed to promote health and longevity. In fermented black tea, polyphenols are converted to phlobaphenes forming aromatic compounds. However, 30–40% of green tea extractable solids are polyphenols known as catechins. The primary catechins in green tea epicatechins (EC), epigallocatechin gallate (ECG), and epigallocatechin (EGC). Other polyphenols present in green tea include flavonoids, flavenol glycosides and depsides such as cholorogenic acid, coumaroylquirtic acid, and a unique acid, theogallin (3-galloyquinic acid). Also present are quinic acids, carotenoids, trigalloyglucose, lignin, protein, chlorophylls, mineral, caffeine and very small amounts of other methylxanthines such as theophylline theobromine and theathine. Green tea has been used in combating various forms of cancer (1–3) such as breast, prostate, esophagus, stomach, pancreas, colon, and various other diseases such as Alzheimer, Parkinson. Green tea has a great effect on enhancing the immune system functioning due to its polyphenol antioxidant characteristics. There is a growing body of evidence that green tea polyphenols are powerful antioxidants that exert their physiological action through combating oxidative stress. Although a large number of publications have appeared that encamp various aspects of green tea physiological activity, understanding of the mechanism is involved in the biological effects of green tea is far from complete. We have focused our study on the Synergistic effect of green tea polyphenols with CoQ9 in rats in order to study antioxidant and free radical scavenging activity which may play a role in lowering LDL-Cholesterol. It may also stimulate detoxifying system such as phase I and phase II metabolic enzymes which increase the formation and excretion of detoxified metabolites of carcinogens. Our results show that a minimal amount of CoQ9 when given in conjunction with green tea extract, could reverse oxidatively induced liver (4) fibrosis with reserpine. The hepatic recovery was assessed through serum hepatic enzymes, namely; GOT and GPT.

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References 1. Afzal, M., Safer, A. M., & Al-Bloushi, S. (2005). CoQ9 potentiates green tea antioxidant activities in Wistar rats. BioFactors, 25, 255– 259. 2. Huang, M.-T., & Ferraro, T. (1992) Phenolic compounds in food and cancer prevention, In M.-T. Huang, C.-T. Ho & C. Lee (Eds.), Phenolic compounds in food and their effects on health II, ACS symposium Series, 507 (pp. 8–34). Washington, DC: American Chemical Society. 3. Oak, M. H., El Bedoui, J., & Schini-Kerth, V. B. (2005). Antiangiogenic properties of natural polyphenols from red wine and green tea. Journal of Nutritional Biochemistry, 16(1), 1–8. 4. Zhou, Y. D., Kim, Y. P., Li, X. C., Baerson, S. R., Agarwal, A. K., Hodges, T. W., Ferreira, D., & Nagle, D. G. (2004). Hypoxia-Inducible Factor-1 Activation by (-)-Epicatechin Gallate: Potential adverse effects of cancer chemoprevention with highdose green tea extracts. Journal of Natural Products, 67(12), 2063–2069.

Antioxidant and Anti MRSA activities of Red Grape Seeds Amar Habib, Esmaeil Saleh and Mohammad Afzal Department of Biological Sciences, Faculty of Science, Kuwait University, Kuwait This study was carried out to evaluate the potential antioxidant and antibacterial activities of grape (Vitis vinifera) pulp and seeds. Samples were analyzed for the determination of total phenols, flavanoids, and anthocyanins. The antioxidant activities of grape extracts were determined using several methods such as DPPH, FRAP, ABTS, and b-carotene/linoleate assay. Antioxidant activity observed in grape seeds was many fold stronger than in grape pulp. This was probably due to higher level of total phenolics present in seeds as compared to grape pulp. Grape pulp and seed extract were also tested for antibacterial activity against 43-strains of MRSA by gel diffusion, growth, and respirometric studies. All MRSA strains were found to be sensitive to grape seed extract. The antimicrobial activity was characterized to be bactericidal targeting synthesis of the bacterial cell wall as shown by scanning and transmission electron microscopy. These valuable activities of grape seeds could be considered important in the treatment of infectious disease caused by MRSA. Key Frontiers in Angiogenesis Modulation: Implications in Vascular Disorders Shaker A. Mousa Albany College of Pharmacy and Pharmaceutical Research Institute, Albany, NY

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The field of angiogenesis modulation is at a major crossroad. Tremendous advancement in basic science in this field is providing an excellent support to the concept with the success of thalidomide in multiple myeloma and the anti-vascular endothelial growth factor in solid tumor when given in conjunction with chemotherapeutic agents. Many attempts are under way to correlate anti-angiogenesis markers to clinical outcome. With regard to angiogenesis stimulation for the treatment of vascular occlusion, wound healing, and other vascular disorders, many novel strategies are being explored at the experimental and early clinical levels. I will discuss the applications of novel nanotechnology approaches in improving the local delivery of newly discovered proangiogenesis agents that initiate new vessel formation and sustain its maturation. This talk will focus on key frontiers in angiogenesis modulation using naturally driven drug targets for the potential treatment of vascular, cancer, inflammatory, and ocular disorders. For Educational Goals: (1) Learn about the role of the different types of pro-angiogenesis factors (2) Learn about the role of the different anti-angiogenesis factors (3) Learn about the proof of the concept in experimental settings (4) Learn about the clinical proof of the concept (5) Learn about the latest advances in pro- and anti-angiogenesis mechanisms (6) Learn about nanotechnology in the delivery of pro- and antiangiogenesis agents

Broad Spectrum Anti-Angiogenic Efficacy of OT-674/ OT-551 in Inhibiting Oxidative Stress and Proagngiogenic Growth Factor-Mediated Angiogenesis Shaker A. Mousa, Donald Armstrong The Pharmaceutical Research Institute (PRI), Albany College of Pharmacy, Albany, NY Purpose: To determine the potential effect of the antioxidant OT-674 and its pro-drug OT-551 on the inhibition of angiogenesis induced by growth factors, cytokine, pro-inflammatory stimuli, and oxidative stress. Methods: The efficacy of OT-674/OT-551 in inhibiting angiogenesis induced by various stimuli, including oxidative stress (H2O2), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), angiotensin II, bradykinin, and endotoxin (LPS) was determined using the chick chorioallantoic membrane (CAM) model of angiogenesis.

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Results: Investigation by our and other laboratories has demonstrated that the pro-angiogenesis effects of bFGF are blocked by monoclonal antibodies directed toward bFGF or the integrin avb3 antagonists and that the pro-angiogenesis effects of VEGF are blocked by specific VEGF antibodies, or other anti-VEGF strategies, in the CAM model. In this current investigation, the potential antiangiogenesis effect of OT-674/OT-551was evaluated in the CAM model, at doses ranging from 30 to 800 lg. Generation of new blood vessels from existing vessels was promoted two- to three-fold by H2O2, bFGF, VEGF, angiotensin II, bradykinin or LPS. The proangiogenesis effect of these different mediators was significantly inhibited (P < 0.01) by OT-674/OT-551 applied topically or by intravenous administration Furthermore, an additive anti-angiogenesis efficacy was demonstrated between OT-674 and avastin or lucentis in inhibiting VEGF-induced angiogenesis. Conclusions: These data indicate the broad potential efficacy of OT-674/OT-551 against the various stimuli used and its potential benefit, either alone or in combination with other single-mechanism-based antiangiogenesis agents such as avastin or lucentis, in the prevention and treatment of angiogenesis associated with diabetic retinopathy, macular degeneration, and other ocular disorders. Antiglycation Study of Some of Natural and Synthetic Compounds Umbreen Khan*, Muhammad Iqbal Choudhary and Atta-ur-Rahman H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan, E-mail: [email protected] Non-enzymatic glycation of proteins or Millard reactions are also called non-enzymatic glycosylation. In human system; this process is related to chronic hyperglycemia, which leads to a series of post-translational modifications of proteins, resulting into late complications of diabetes. Diabetes mellitus is ubiquitous endocrine disorder, exemplifies by hyperglycemia and predisposes patients to of unremitting complications affecting the eyes, blood vessels, nerves, and kidneys. Glycation is the result of the bonding of sugar molecules, such as fructose or glucose, to a protein or lipid molecule, without the controlling action of an enzyme.

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All blood sugars are reducing molecules. Glycation may occur either inside (endogenous) or outside (exogenous) of the body. In our study, crude extracts of a number of medicinal plants, fruits, vegetables, and spices, as well as several pure natural and synthetic compounds, were screened for their potential antiglycation activity. Several of them have shown interesting antiglycation activity in an in vitro protein glycation model assay. References 1. Ahmed, N. (2004). Advance glycation endproducts-role in pathology of diabetic complications. Journal of Diabeties Research and Clinical Practice, 67, 3–21. 2. Nakagawa, T., Yokozawa, T., Terasawa, K., Shu, S., & Juneja, L. R. (2002). Protective activity of green tea against frees radical- and glucose-mediated protein damage. Journal of Agricultural and Food Chemistry, 50, 2418–2422.

Protective Role of Garlic Against Oxidative Stress in Hypertension Hana Drobiova, Muslim Ali, Khalid Al-Qattan, Martha M. Thomson Department of Biological Sciences, Faculty of science, Kuwait University, Kuwait Hypertension is a common problem in developing countries. It may lead to development of degenerative diseases if untreated. It was shown that reactive oxygen species (ROS) may play a role in the pathobiology of hypertension. Garlic (Allium sativum) contains active ingredients (allicin) that may act as antioxidants. Therefore, garlic may be used as a treatment. This study was performed to assess the relationship between the hypotensive effects of garlic and total antioxidant levels in plasma of 2 kidney-1-clip (2K-1C) hypertensive rats. Rats were operated to induce 2K-1C model of hypertension. After 1 week, they were divided into two groups (n = 5). One group was not treated (control), and the other group was treated with garlic extract intraperitoneally on daily basis for 3 weeks. Blood was drawn, and blood pressure measured after 1, 2, and 3 weeks of treatment. Blood plasma was analyzed for antioxidant levels. It was demonstrated that garlic reduces blood pressure in hypertensive rats by 50% after 3 weeks of treatment. In addition, the level of antioxidants in hypertensive rats increased due to garlic treatment. This increase in antioxidant levels may contribute to the reduction of hypertension.

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Anti-Oxidants from Green Tea and Pomegranate for Chemoprevention of Prostate Cancer Hasan Mukhtar Helfaer Professor of Cancer Research, Director and Vice-Chair for Research, Department of Dermatology, University of Wisconsin, 1300 University Avenue, Medical Sciences Center, B-25, Madison, WI-53706, USA, Phone:608-263-3927, Fax:608-263-5223, E-mail:[email protected] Among males, prostate cancer is the second leading cause of cancer-related deaths in North America with similar trends in many Western and developing countries. One way to control prostate cancer is through chemoprevention, which refers to the administration of synthetic or naturally occurring agents to block, reverse or delay the process of carcinogenesis. For two major reasons prostate cancer is an ideal candidate disease for chemopreventive intervention. First, it is a unique malignancy which generally grows very slowly, likely for decades, before symptoms arise and a diagnosis is finally established. Second, because of a long latency period it is typically diagnosed in men >50 years of age. For a variety of reasons, the most important of which is human acceptance, for chemopreventive intervention naturally occurring diet-based agents are preferred. Most chemopreventive agents are antioxidant in nature. We have been defining the usefulness of dietary antioxidants for chemoprevention of prostate and other cancers. This presentation will focus on prostate cancer chemopreventive effects of polyphenolic anti-oxidants from green tea and pomegranate. Utilizing an autochthonous mouse model of prostate cancer we have shown that oral infusion of a polyphenolic fraction isolated from green tea at a human achievable dose, equivalent to six cups of green tea per day, resulted in significant delay in primary tumor incidence and tumor burden, significant decrease in prostate and genitourinary weight and increased overall survival. The striking observation of this study was that green tea infusion resulted in almost complete inhibition of distant site metastases. Oral administration of pomegranate fruit extract to athymic nude mice implanted with androgen-sensitive CWR22Rm1 cells resulted in a significant inhibition in tumor growth concomitant with a significant decrease in serum prostate-specific antigen levels. These results have recently been corroborated in clinical trials where cancer incidence was only 3% in volunteers that consumed green tea compared to 30% on placebo. A phase II clinical trial for men with rising PSA treated with 8

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ounces of pomegranate juice daily suggested a statistically significant prolongation of PSA doubling time. It is envisioned that some of these dietary anti-oxidants known to have potential utility in cancer prevention and therapy could be custom tailored as cocktails in concentrations that can easily be consumed by humans for delaying prostate cancer.

An Electrochemical Device for Monitoring Antioxidative Property of Suspected Antioxidants M. Mohammad, M.Tahir Soomro, and M. Tariq Reactive Intermediates-Free Radical Chemistry Group, International Center for Chemical and Biological Sciences, H.E.J Research Institute of Chemistry, University of Karachi, Pakistan, Karachi-75270, Email: [email protected]; [email protected] An antioxidant is a chemical which reacts with (or scavenges) the reduced molecular oxygen species (superoxide, hydroxyl radical etc.) It is of great interest to the general public, medical and nutritional experts, and health and food science researchers to know the antioxidant capacity and constituent in the foods we consume. Due to the complexity of the composition of foods, separating each antioxidant compound and studying it individually is costly and time consuming or inefficient. Therefore, it is very appealing to researchers to have a convenient method for the quick quantitation/ monitoring of antioxidant’s effectiveness. In this article, an electrochemical monitoring device to test antioxidative property of suspected antioxidants is reported. The test has been carried out on several known compounds such as vitamin C, uric acid, vitamin E, Butylated hudroxyl Anisolt (BHA), Butylated hydroxyl Toulene (BHT), Benzoquinone, as well as on extracts of some substance in common use such as Miswak (siwak), Clove (syzygium aromaticum), saffron (crocus sativus), and harsinghar (nycanthes arbortrisis) flower stem etc. The electrochemical device was based on the reversible electrochemical reduction of oxygen in an aprotic solvent in the absence and in the presence of a suspected antioxidant. The device consists of a three electrode assembly (a working electrode, a reference electrode, and a counter electrode) in single pin (rod) configuration for rapid measurement. The solvent was DMSO (HPLC grade dried over molecular sieve) and supporting electrolyte was Tetrabutyl ammounium Iodide. The method was found to give reproducible results and is rapid.

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Functional Analysis of ASK2 in Two-stage Skin Tumorigenesis Takayuki Iriyama, Hideyuki Kinoshita, Kohsuke Takeda, and Hidenori Ichijo Cell Signaling, Grad. Sch. Pharmaceut. Sci., University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan, 113– 0033 Apoptosis signal-regulating kinase 2 (ASK2; also known as MAP3K6) is a recently identified MAP3K in the JNK and p38 MAP kinase pathways that is highly related to ASK1. ASK2 forms a heteromeric complex with ASK1 and mediates various stress responses. However, physiological functions of ASK2 have been largely unknown. Since ASK2 is highly expressed in mouse skin, we investigated the susceptibility of ASK2deficient (ASK2–/–) mice to skin tumorigenesis. In the two-stage skin tumorigenesis experiment using an initiator DMBA and a tumor promoter TPA, papillomas were induced earlier and more in ASK2–/– mice than in wild type (WT) mice. DMBA-induced apoptosis of the skin keratinocytes was inhibited in ASK2–/– mice. Moreover, primary keratinocytes derived from ASK2–/– mouse skin were refractory to DMBAinduced p38 activation and apoptosis. These results suggest that ASK2 is required for DMBA-induced apoptosis and play a pivotal role in tumor suppression. This research was supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Sciences and Culture of Japan, CREST, Japan Science and Technology Corporation, and Strategic Approach to Drug Discovery and Development in Pharmaceutical Sciences, Center of Excellence (COE) Program.

A Double Mutant of Tomato Lycopersicon esculentum with High Pigments Content Arif S. A. Alhammadi Department of Life Sciences, Faculty of Sciences, University of Sana’a, Sana’a, Republic of Yemen, [email protected] Epidemiological evidences suggest a strong correlation between increased consumption of tomatoes and tomato products and the prevention of chronic diseases such as cancer and cardiovascular diseases. Lycopene, an antioxidant present in tomatoes has been identified as being responsible for the beneficial effects of tomatoes. The high pigment (hp) mutant characterized by dark green fruit when it is immature and deep red

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mature fruit is a monogenic mutant of tomato was crossed with atroviolacea (atv) monogenic nonallelic mutant which have the same phenotype. Although it was not easy to distinguish the hp and atv segregating mutants in the F2 generation the confirmation of the hpatv double mutant was done genetically. The double mutant showed darker phenotype in stem leaves and much darker fruits with black islands due to the higher pigments content. In addition to the higher pigments content which is of great nutritional value, the high pigmentation gives the plants higher resistance to the stresses. The quantitative differences in pigments of the wild type, single and the constricted double mutants will be presented. Stabilisation of Refined Olive Oils with Natural Antioxidants Recovered from Chemlali Olive Leaves Extract Mohamed Bouaziz, Ines Fki, Hedya Jemai & Sami Sayadi Laboratoire des Bioproce´de´s, Centre de Biotechnologie de Sfax (CBS), BP: « K », 3038, Sfax, Tunisia Antioxidants are major ingredients which protect the quality of foods by retarding oxidation. In the edible oil industry, synthetic antioxidants are often used because they are effective and inexpensive. However, increased popularity of natural food additives may prompt more food manufacturers to replace synthetic antioxidants with ingredients containing natural antioxidative compounds. Therefore, research on natural ingredients has gained momentum as they are generally considered to pose no health risk to consumers. Olive leaves might be a natural source of useful substances. In this study, we have identified the main phenolic compounds present in Olea europaea. L leaves extracts of Chemlali cultivar before and after enzymatic hydrolysis. Results revealed that oleuropein was the abundant product in leaves extract, where hydroxytyrosol was the major compound in the hydrolysate leave extract. The antioxidant activity of extract and its hydrolysis was examined in vitro in lipid medium. The two extracts were added to refined and husk olive oils and their effectiveness was compared with that of a-tocopherol and a control at 50C. The stability of oils was determined by measuring peroxide value (PV). Oils treated with hydrolysate extract exhibited the lowest PV value as compared with the control sample. Moreover, the addition of leave extract and a-tocopherol at 400 ppm had similar effects in retarding oxidation of refined oils. The results suggested that olive leaves hydrolysate

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which is rich in hydroxytyrosol, possess useful antioxidant properties and may become important in the search for natural replacement for synthetic antioxidant food additives. Free Radical Injury for in vivo Induction of Platelet Aggregation: A Model in Microcirculatory Studies of the Brain Farouk El-Sabban Department of Family Sciences, College for Women, Kuwait University, Kuwait This model involves the induction of in vivo platelet aggregation (thrombus formation) in brain microvessels of laboratory animals. The photochemical injury is caused by a generated free radical (singlet oxygen) through a direct exposure to intense mercury light on vessels in which a dye (sodium fluorescein) circulates. This technique utilizes a microscope-closed circuitry television setup for continuous monitoring of occurring events in vessels and for recordings on video-tapes, for further viewing and analysis. The generated free radical injures the vascular endothelium, to which platelets aggregate in response. The times for both the first observed platelet aggregate and for platelets to grow and block the vessel are recorded by a set of four stopwatches. This in vivo animal model has the advantage over the in vitro method, as all interacting factors are present. Thus, influences on platelet aggregation (whether anti- or pro-aggregatory) can be studied under well-controlled conditions. This method was employed in assessing the influence of various factors on thrombotic processes, such as: drugs, natural products, medicinal plants—as well as those which are environmental and nutritional. Additionally, this free radicalgenerating biological insult was also used as a model for stroke and has been utilized in inducing synaptic dysfunction in neuronal tissue. The employment of this technique in different types of biologically-oriented experimental work can be further expanded.

Protective role of Curcum on CCl4-induce Oxidative Stress On Liver and T-lymphocyte Subpopulations in Wistar Rats Hana’a Abu-Rizk, M. Hisham Mansour and M. Afzal Department of Biological Sciences, Faculty of science, Kuwait University, Kuwait

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Curcuma longa herb is one of the most commonly used spice with strong antioxidant and potential hepatoimmuno protective activities. In the present study antioxidant effect of curcum crude extract was compared with pure curcumin, a component of curcum, in carbon tetrachloride (CCl4) induced oxidative-stress in male Wistar rats. A 0.2% aqueous solution of CCl4 was administrated by gavage to animals for one week at a daily dose of 2 ml/213.67 g average body weight. CCl4 caused a significant structural damage to hepatocytes, in addition to a significant elevation in serum glutamicpyruvic transaminase and serum glutamic oxaloacetic tranaminase (SGPT, SGOT, respectively). A significant elevation in liver TBARS was also observed. Further more, CCl4 showed a selective and significant effect on immature (PNA+) thymocytes and peripheral helper (CD4+) T lymphocytes in spleen. This resulted in a significant reduction in CD25, CD71, and Con A receptor expression. Treatment with curcum crude extract at two different doses (single, double doses) and the double dose of curcumin, all showed a significant structural recovery among hepatocytes along with a significant reduction in the level of GPT, GOT, and TBARS. A prominent restoration in the viability and expression of CD25, CD71 and Con A receptors among immature (PNA+) thymocytes, and splenic helper (CD4+) T lymphocytes was seen. Effect of curcum crude extract and the pure fraction (curcumin) were compared at both doses and curcum crude extract at double dose was found to be more effective. Nitric Oxide Radical Scavenging Activity of Some Compounds-Indicators of Potential Antioxidant Activity Iman Omar, M. Iqbal Choudhary, and Atta-urRahman H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi- 75270, Pakistan, Ph.: 111222292(ext.-217), E-mail: [email protected] Free radicals play an important role in carcinogenesis through their involvement in breaking of DNA strands. Numerous pathological events are associated with the generation of reactive oxygen species (ROS) constituting a key mechanism of tissue injury. Free radicals can react with vital cellular components such as nucleic acids, proteins, and cell membranes thus damaging the cells. Nitric oxide radical has recently been recognized as an important messenger molecule and has a broad

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spectrum of functions in biological systems, ranging from physiological control to pathological effects. Nitric oxide radicals are generated in the biological system by the action of an enzyme called Nitric oxide synthase on an amino acid called L-arginine. Over production of nitric oxide in inflammatory tissues under chronic conditions by the inflammatory cascades can damage DNA, RNA, lipids, and protein functions leading to increased mutations and altered enzymes which in turn lead to carcinogenesis. Antioxidants are the most promising candidates in modern research for the prevention of different diseases and for promotion of general healthcare. For the discovery of nitric oxide radical’s inhibitors, various classes of compounds were assessed for their nitric oxide radical scavenging potential by using a high-throughput mechanism based assay. Series of synthetic vanadium complexes, and alkaloids such as imidazolones and indoles showed significant results. The compounds of exciting results will be presented in this presentation. References 1. Li, C.-Q., & Wogen, G. N. (2005). Cancer Letters, 226, 1–15. 2. Badami, S., Gupta, M. K., & Suresh, B. (2003). Journal of Ethanopharmacology, 85, 227–230.

Food: A Source of Antioxidants Reshod A. Al-Shagrawi Prof. of Nutrition, Food and Nutrition Dept. King Sau’d Unversity, Riyadh, Saudi Arabia, P.O. Box 2460 KSA. Riyadh, e-mail : [email protected], Fax no. 4632163 Millions of vital processes occurring in every seconds by the different kinds of body cells including Digestive, Respiratory, and Circulatory Systems as well as liver and kidney functions, the out come of these vital processes what is called the free radicals as a results of different biochemical reactions happened, the accumulation of these free-radicals could be one of the important source of carcinogen that could lead to cancer of different part of the body the body causing overloading on the liver, kidney to get rid of these toxins. To help free radicals reduce the effect of these freeradicals in the body need antioxidant. So the importance of antioxidants is getting rid of those toxins to keep the body vital system in a good condition all the time. The most important types of those anti oxidant is the vitamins such as E, C, A, and some trace elements like zinc, selenium, xeronine, cobalt, and chromium, its

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sources variable we can obtain from fresh vegetables, fruits or by some medical and nutritional supplements under medical supervision to help regular cleaning of different body system. In this article I will present the different type of food source of this antioxidant coming food source and how to improve these functions and what factor can effect the bioavailability. In Vitro Reaction of Bromine Atom Free Radical with Aminoacid and DNA Components M. Mohammad, M. Tariq, and M.Tahir Soomro Reactive Intermediates-Free Radical Chemistry Group, International Center for Chemical &Biological Sciences, H.E.J Research Institute of Chemistry, University of Karachi, Pakistan, Karachi-75270, Email: [email protected] In order to study the damaging or beneficial properties of bromine atom free radical, reaction of the free radical (BrÆ) with some biologically important compound were investigated. BrÆ was generated through electrochemical oxidation of bromide ion (Br). The free radical (BrÆ) was monitored using cyclic voltammetry and spectroelectrochemistry. Through this technique it was ascertained that the product of electrochemical oxidation of Br– (bromide ion) was BrÆ (bromine atom free radical). It was also ascertained that the substrate underwent reaction with BrÆ and not with dibromine (Br2), a dimer product of BrÆ. It was found that compound glycine and uracil underwent catalytic reaction, while adenine and 2-picoline reacted with BrÆ as simple chemical reactions. Compounds like ethanol, sucrose, glycerol, and acetic acid did not show any reaction at the time scale of cyclic voltammetry (50 mV/s scan) Tackling Free Radical Ailment among Lagosian: The Affordable Option Wahab Abdulfatai Olabamidele, Adeyemi Ebenezer Olusegun 54 Alhaji Jinadu Street, Ijeshatedo, Lagos 101015, Nigeria, Fax: +16106438199, E-Mail: [email protected] Free radical are chemically active because of their unpaired electron. Though, they occur in low concentration naturally in the body. However certain conditions and lifestyles shoot up their concentration leading

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to some major medical malfunctions. Aging and degenerative diseases as well as an open ending list are either initiated or complicated by free radicals. Acne, cancer, cold and flu, fatigue, infertilities, allergies, arthritis, rheumatism and even stress are usually aggravated if not initiated by free radicals. They are kept under control by the use of antioxidants, a group of chemicals (reducing agent) that can attack and block the chain of reaction of free radicals in the body. Lagos with its highest population density in Africa is 100% metropolitan and congested filled with several millions of Nigerians battling poverty and general insufficiency. In addition to this is the Tropical climate, which is also highly humid in a country where environmental conservation and pollution control are attended to only with lip-serve by successful administrations. These unfortunate conditions, which are the most favorable for free radicals medical problems, Had led to highly level of the prevalence of practically all ailment, both minor and degenerative among Lagosians. High morality, low life expectancy as well as poor well being among the Lagos populace are glaring. Antioxidants, which used to be exclusively products of orthodox medicine, had been unreachable and affordable to majority of the poverty-ridden populace of this poor African city. However, the discovery of several local sources of formidable antioxidants both as dietary/food supplement and herbal medicine by Nigerian researchers as turned around the fate of Lagosians. This presentation examines efforts that could be used to elevate the medical situations of the most vulnerable strata of the Lagos populace via a sustainable mass production of ‘‘Jobelyn’’ a most powerful natural antioxidant recently certified as the best natural antioxidant known to Man after series of efforts by Herbal and Orthodox medical researchers based in the University of Lagos and Benin, both in Southern Nigeria. Regulatory Mechanisms of ASK1 Activation by Ubiquitination Hiroaki Nagai, Takuya Noguchi, Kohsuke Takeda, Hidenori Ichijo Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan, 113–0033 Apoptosis signal-regulating Kinase 1 (ASK1) is a mitogen-activated protein (MAP) kinase kinase kinase that activates the p38 MAP kinase and c-Jun

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N-terminal kinase (JNK) signaling pathways. ASK1 is activated by various stresses such as oxidative stress and ER stress. ASK1 plays crucial roles in stress-responses in vivo by regulating a large variety of cellular functions including apoptosis. We previously demonstrated that ASK1 constitutively forms a high molecular mass complex, and oxidative stress induces higher molecular mass complex formation at least in part by recruiting TRAF family members to ASK1 complex. However, it has been predicted that unidentified components may further be recruited to the ASK1 complex under oxidative stress conditions. Affinity purification of the ASK1 complex by using overexpression system in HEK293 cells revealed that a deubiquitinating enzyme is recruited to the ASK1 complex in an oxidative stress-dependent manner. The deubiquitinating enzyme possesses deubiquitinating enzyme activity for both K48- and K63-linked ubiquitination in vitro. On the other hands, we found that ASK1 is ubiquitinated in an oxidative stress-dependent manner. Taken together, ASK1 activation appears to be regulated by its ubiquitination and deubiquitination. Regulatory mechanisms of ASK1 by ubiquitination and their potential roles in stress response will be discussed. This research was supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Sciences and Culture of Japan, CREST, Japan Science and Technology Corporation, and Strategic Approach to Drug Discovery and Development in Pharmaceutical Sciences, Center of Excellence (COE) Program. ASK Family Proteins in Stress Response and Disease Hidenori Ichijo Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo & CREST, 7-3-1 Bunkyo-ku, Tokyo, Japan, 113–0033 Actions of pro- and anti-apoptotic factors are often modulated by phosphorylation and dephosphorylation, and protein kinases and protein phosphatases thus contribute to the regulation of cell death decisions made in response to various stresses. Apoptosis Signalregulating Kinase 1 (ASK1) is a member of the mitogenactivated protein (MAP) kinase kinase kinase family, which activates both the MKK4/MKK7-JNK and MKK3/MKK6-p38 MAP kinase pathways and constitutes a pivotal signaling pathway in various types of stress responses [1]. ASK1 plays pivotal roles in oxidative stress [2]- and endoplasmic reticulum (ER) stress [3]-induced apoptosis and calcium signaling [4] that are

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implicated in the pathophysiology of a broad range of human diseases. Moreover, ASK1-p38 pathway was recently found to play important roles in the innate immune responses [5]. LPS induced the production of intracellular reactive oxygen species (ROS), which is specifically required for LPS-dependent formation of the TRAF6-ASK1 complex and subsequent activation of the ASK1-p38 pathway. Our data demonstrate that ROS-dependent TRAF6-ASK1-p38 axis is crucial for LPS-induced innate immune responses. In this symposium, I will review our recent findings on the redox control [6] and pathophysiological roles of ASK1 in stress responses. I will summarize the roles of stressactivated MAP kinase pathways and update our new findings on the pathophysiological roles of ASK family proteins in stress responses. Molecular mechanisms by which ASK family proteins determines cell fate such as differentiation and apoptosis will also be discussed. References 1. 2. 3. 4. 5.

Ichijo, H. et al. (1997). Science, 275, 90–94. Saitoh, M. et al. (1998). TheEMBO Journal, 17, 2596–2606. Nishitoh, H. et al. (2002). Genes Development, 16, 1345–1355. Takeda, K. et al. (2004). EMBO Reports, 5, 161–166. Matsuzawa, A., Saegusa, K. et al. (2005). Nature Immunology, 6, 587–592. 6. Noguchi, T. et al. (2005). Journal of Biological Chemistry,280, 37033–37040.

Dietary Ginger Impact on Hepatic Antioxidant Enzyme Homeostasis under Ethanol Induced Free radical Stress Conditions in Albino Rats

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treatment. The second group of rats received 2.0 g of ethanol post orally and the third group of rats received 1% of dietary ginger through diet for a period of 4 weeks. The last group of rats received both alcohol and ginger as described in group II and group III. After completion of last treatment the selected antioxidant enzymes such as Superoxide dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GSH-Px) and Glutathione Reductase (GR) were estimated in the liver tissue. The activities of these antioxidant enzymes were significantly decreased in ethanol treated group, but the same parameters were augmented with ginger feeding in group III when compared to their sedentary control rats. Nevertheless, the activities of these antioxidant enzymes were significantly elevated with combination treatment in group IV which received both alcohol and ginger. The decrease of SOD was 32% and CAT was 25% with ethanol intoxication. The increased SOD and CAT with ginger treatment was 39 and 47% respectively. Among these antioxidant enzymes, GR showed the maximum percent decrease (55%) with ethanol treatment and remarkable increase (65%) with combination treatment compared to that of their SC group. The present findings reveal that over production of free radicals with ethanol consumption may be countered by the dietary ginger supplementation as evidenced by improved antioxidant status in the hepatic tissue. Hence, these findings suggest that treatment of ginger to alcoholics may have protection against the ethanol induced free radical toxicity.

Mallikarjuna, K., Sahitya Chetan, P., Rajendra, W., and Sathyavelu Reddy, K.

Green and Black Tea: The New Era of Antioxidants and Free Radical Scavengers

Division of Molecular Biology, Department of Zoology, Sri Venkateswara University, Tirupati 517 502, A.P., India, Mail id: [email protected], [email protected]

Muslim Ali, Amina Jassim Haider, Rasmia Al-Sous, Martha Thomson, Khaled K. Al-Qattan and Hana Drobiova

It is well known that intake of ethanol enhances the production of free radicals during its own metabolism and disturb the hepatic antioxidant homeostasis, finally leads to cell death. From the ancient times dietary ginger is widely used as an antioxidant in Ayurvedic medicine in India. In view of antioxidant property of ginger the current investigation has been conducted to investigate the influence of ginger on hepatic antioxidant enzyme system in alcoholics. Wistar strain male albino rats (170 ± 10 g) were divided into four groups such as group I—Sedentary Control (SC), group II—Ethanol treated (Et), group III—Ginger treated (Gt) and group IV—Ethanol + Ginger (Et + Gt)

Department of Biological Sciences, Faculty of Science, Kuwait University, P.O.Box 5969, 13060-Safat, Kuwait There have been a number of reports that drinking tea is healthier than water. Experts believe flavonoids in tea are the key ingredients in promoting health. Generally three to four cups of tea are consumed daily. There is a certain percentage of the population that consumes in excess of four cups of tea per day. No reports, however, are available on the antioxidants levels and toxicity from drinking excess amounts of tea. In the present study, we would like to report results from excess tea consumption on the total antioxidant levels and its effect on the cardiac enzymes and other parameters.

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In the present experiment, 40 male weanling Sprague–Dawley rats were divided into four groups. Group I were on water, Group II were on black tea extract, Group III were on green tea extract and Group IV were on drinking water enhanced with vitamin C. Their weights and amounts of fluids consumed were recorded. After 7 weeks the rats were sacrificed after overnight fasting and the blood was collected by cardiac puncture. The serum was obtained by clotting the blood at room temperature and the total antioxidants levels, serum protein, glucose, lipids, and cardiac enzymes were measured. The results showed a significant decrease in the mean weights of the rats in the vitamin C groups compared to the control group. The total antioxidants levels were significantly increased in all the treatment groups compared to the control group. A highly significant increase in the black tea group was also observed. The increase in the total antioxidants levels was corroborated with increase in plasma protein. A significant increase in blood glucose level was only observed in the group on black tea. No significant changes in blood glucose, lipids, and enzymes were observed in treated groups compared to the control group. Our results show that an increase in total antioxidants levels by consuming tea could be an important factor in the protection from major chronic diseases. The results also suggest that drinking tea in large amounts causes no major toxicity in the body. Acknowledgements: This work was partly supported by a grant provided by the College graduate Studies, Kuwait University, Kuwait. Involvement of ROS-dependent activation of ASK1-p38 pathway in ATP-induced apoptosis in macrophage Takuya Noguchi, Shiori Murakami, Hisashi Fukutomi, Kohsuke Takeda, and Hidenori Ichijo Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan, 113–0033 Extracellular ATP, an autocrine or paracrine intercellular transmitter, is known to induce apoptosis in macrophages. However, the precise signaling mechanisms of ATP-induced apoptosis remain to be elucidated. Here we showed that activation of p38 mitogenactivated protein kinase (MAPK) plays a critical role in ATP-induced apoptosis. p38 activation and apoptosis in macrophages were induced by ATP. ATP-induced apoptosis was mediated by production of reactive oxygen species (ROS) from the mitochondria. Furthermore,

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ATP-induced ROS generation, p38 activation, and apoptosis were almost completely inhibited by selective P2X7 receptor antagonists or P2X7 receptor knockdown. We also found that ATP-induced apoptosis were diminished in ASK1-deficient macrophages accompanied by the lack of p38 activation. These results demonstrate that ROS-mediated activation of ASK1-p38 MAPK pathway downstream of P2X7 receptor is required for ATP-induced apoptosis in macrophages. This research was supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Sciences and Culture of Japan, CREST, JapanScience and Technology Corporation, and Strategic Approach to Drug Discovery and Development in Pharmaceutical Sciences, Center of Excellence (COE) Program. Bi-directional Role of Oxidative Stress in Sperm Capacitation by Glucose Omu AE1, Al-Azemi MK1 Kehinde E2, Omu FE3, Mathew C4, Fatinikun T1, Anim JT5 Departments of Obstetrics and Gynaecology1, and Surgery2, Anatomy4 and Pathology5, Faculty of Medicine, Kuwait University, and College of Nursing, PAAET3, Kuwait Glucose increases sperm capacitation but the mechanism has not been elucidated. Objective of the study is to evaluate the role of lipid peroxidation in sperm capacitation with glucose and the effect of antioxidants. Patients and Methods: About 20 men with infertility, but with normal sperm parameters and normal blood glucose levels were evaluated with semen analysis, fasting blood and seminal glucose; glucose concentrations of 0, 2.5, 5, 7.5, 10, 12.5, and 15 mM in RPMI. (1) plus 100,000 sperm to each concentration and incubated for 1 h at 37C and (2) glucose vitamin E. Semen analysis, MDA and Acridine Orange denaturation and Electron Microscopy.(EM) of Sperm. Results: Blood glucose concentration was 4.4 ± 0.8 (95% CI; 3.8, 4.9) but not detectable in semen. Sperm concentration 98 · 105/ml; Progressive motility 46 ± 11%, Sperm immotility 15 ± 6% and Blood MDA 2.3 ± 0.4 nmol/ml and seminal plasma MDA 2.4 ± 0.3 nmol/ml. Sperm motility increased by 20% at 7.5 mM glucose concentration and thereafter declined from 10 to 15 mM by 30%. MDA level increased from 1.5 ± 0.4 nmol/ml at glucose level of 0 mM to 4.5 ± 1.2 nmol at 15 mM glucose concentration.

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Sperm DNA fragmentation Index (DFI) increased by 23%. Electron microscopy showed increased disruption of the acrosome and midpiece. Most of the abnormalities of MDA, sperm motility, DFI, and ET were significantly reduced by vit E. Conclusion: High levels of glucose leads to increased lipid peroxidation and sperm damage and vit E, reduced the effect of oxidative stress on sperm lipid peroxidation. Correspondence: Dr. Alexander E. Omu, Department of Obstetrics and Gynaecology, Faculty of Medicine, HSC, Kuwait University, PO Box 24923 Safat, 13110 KUWAIT. Telephone: 5319601, Fax 5338906 and e-mail: [email protected]

Furthermore, lipid peroxidation using bovine brain phospholipid (Fe+3/ascorbate) and rat liver microsomes (Fe+3/ADP/NADPH) were also analyzed. Hexane (IC50 = 30 lg/ml) and ethyl acetate (IC50 = 6.0 lg/ ml) extracts inhibited lipid peroxidation in both the systems while methanol extract was active only in Fe+3/ ascorbate system (IC50 = 28.0 lg/ml). Conclusion: A. corniculatum derived extracts possesses free radicals (ÆOH, O
2 ) scavenging and anti-lipid peroxidation properties that may be related to its antiinflammatory activity.

Anti-oxidant Activity in Aegiceras corniculatum (Stem): A Possible Mechanism Against Inflammation

Hina Siddiqui, M. Iqbal Choudhary, and Atta-urRahman

Talat Roome, Ahsana Dar, Sabira Naqvi, Shamsher Ali and M. Iqbal Choudhary

H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi- 75270, Pakistan, Ph.: 111222292(ext.-217), E.mail: [email protected]

International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan, Fax No.: 4819018. E-mail: [email protected]; [email protected]; [email protected] Aegiceras corniculatum, a mangrove predominantly found in Indo-Pacific region including Pakistan. Traditionally it is used against variety of inflammatory diseases. Different chemical constituents have been isolated, notably benzoquinone, coumarins, flavonoids, pentacyclic triterpenes, and polyphenols. It is well established
that free radicals such as superoxide anions O
2 , hydroxyl radicals (ÆOH) and peroxy are generated during inflammation and infection. We previously reported anti-inflammatory activity in this plant. In the present study, inflammation was induced in mice (NMRI) using glucose oxidase and paw weight was noted. At 100 mg/kg, ethyl acetate and methanol extracts caused 60%, whereas hexane extract caused only 30% reduction in paw oedema. These results showed that the anti-inflammatory activity may be related to the free radical (ÆOH) scavenging potential of this plant. Human neutrophils were used to generate O
2 by phorbol-12-myristate-13-acetate (PMA) via NADPH oxidase complex. The radical generation was measured spectrophotometrically by the reduction of sulfonated tetrazolium salt WST-1. Methanol extract was inactive while hexane (IC50 = 3.7 ± 0.67 lg/ml) and ethyl acetate (IC50 = 8.4 ± 0.67 lg/ml) suppressed the O
2 generation in a dose dependent manner.

Antioxidants: Miraculous Substances for Longivity and Health

Free radicals are highly reactive species that are generated in the body during normal metabolic functions or introduced from the environment. They are inherently unstable, as they contain ‘‘extra’’ energy. To reduce their energy load, free radicals react with certain chemicals in the body and in this process, interfere with the cell’s ability to function normally. Antioxidants work in several ways; they may reduce the energy of the free radicals, stop the free radical from forming in the first place, or interrupt an oxidizing chain reaction to minimize the damage caused by free radicals. Free radicals are believed to play a role in more than 60 different health problems, including the aging, cancer, and atherosclerosis. Reducing exposure to free radicals and increasing intake of antioxidant, nutrients has the potential to reduce the risk of free radical-related health disorders. There is an increasing interest in antioxidants, particularly with reference to the prevention of the presumed deleterious effects of free radicals in the human body. Therefore a large number of compounds of natural and synthetic origins were screened. Certain classes of natural and synthetic compounds such as flavonoids, coumarins, heterocyclic compounds, and vanadium complexes were found to be significantly active against DPPH radical scavenging assay and TBARS assay. Few interesting results of our ongoing research will be presented in this presentation.

Mol Biotechnol (2007) 37:81–97

References 1. Molyneux, P. (2004). Songklanakarin Journal of Science Technology,26(2) 2. Shaheen, F., Ahmad, M., Tareq, M., Jalil, S., Ejaz, A., Sultankhodjaev, M., Arfan, M., Iqbal Choudhary, M., & Rahman, A. (2005). Phytochemistry, 66 935–940.

Evaluation of Green Tea Minerals by Elemental X-ray Microanalysis A. M. Safar, M. Afzal, S. Al-Bloushi, and M. Rafique Department of Biological Sciences, Faculty of Science, Kuwait University, Kuwait Green and black tea are both members of Theaceae family. Since antiquity, both forms of tea are commonly used as cold or hot beverages. Green tea is particularly rich in catechins, and its consumption is invariably linked to promoting health and longevity, the benefits which black tea, with its phlobaphenes content may not provide. Our earlier study on green tea extract, using animal model, showed a reversal of damaging oxidative stress induced by reserpine in rat hepatocytes, as demonstrated by electron microscopy and biochemical studies. In addition, when consumed by rats, green tea polyphenols exhibited synergism with CoQ9, providing better protection against oxidative stress, and lowering LDL-cholesterol. The composition of green tea tends to vary with season, and also its cultivation in different geographical regions of the world. In the present study, we analyzed five different samples of green tea for their mineral content, and compared the results with those obtained from regular black tea. All samples were dried to a constant weight, crushed, fixed on carbon stubs, and loaded in Jeol-JSM-6300 SEM. Elemental X-ray microanalysis for Fe, Mg, P, S, Cl, and K were carried out at 20 kV using Rontec’s (Germany) Edwin Energy Dispersive Spectroscope (EDS). Si(Li), LN2 dewar detector of 127 eV resolution was used. Samples were analyzed for Fe, Mg, P, S, Cl, and K in a given area by electron beam (3–4 lm2). Among inorganic elements, K level range (0.58–2.17 w/w%) was found to be highest in all the samples analyzed and one of the sample had 4.96 w/w% of K. These results clearly showed Potassium to be the key element in green tea, indicating that, among other factors, potassium probably was the major player in health benefits of green tea. Our results demonstrate the significance of K in cell vitality, suggesting that its overall health benefits in aging process cannot be overlooked. Among other things, K is important in the transmission of nerve impulses, the contraction of cardiac, skeletal, and

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smooth muscle, the production of energy, the synthesis of nucleic acids, the maintenance of intracellular tonicity and the maintenance of normal blood pressure. It is known that maintaining a dietary sodium-to-potassium ratio of at least 1:4 can protect against hypertension, strokes and premature death. Epidemiological and clinical studies have shown that potassium intake has an important role in regulating blood pressure in both the general population and people with high blood pressure. High potassium intake may have other beneficial effects independent of its effect on blood pressure. Green tea is blessed with high potassium level that can be very beneficial for its consumers. Further studies need to be undertaken to substantiate these findings as regards green tea’s propensity in enhancing health. Total Antioxidant Status, Nitric Oxide and Endothelin-1 in Serum and Tears of Patients with Cataract Hager E. Amer1, Atef M. Ibrahim1, Rawhia A. Khalifa2, and Mohamed M. El-Mattarawy3 1

Biochemistry Department - Research Institute of Ophthalmology. Associate Professor In Faculty of GirlsSaudi Arabia. 2 Clinical Pathology Department Research Institute of Ophthalmology. 3 Ophthalmic Department - Research Institute of Ophthalmology Purpose: Oxidative damage to lens proteins is a major factor leading to cataract formation. This study was undertaken to assess the relation between different types of cataract and levels of total antioxidant status, nitric, and endothelin-1 in serum and tears. Methods: Serum and tears samples were collected from 70 patients with, senile cataract, diabetic Cataract, Congenital Cataract, and Cataract associated with glaucoma and 15 subjects who served as control subjects. Total antioxidant status, nitric oxide and endothelin-1 were carried out to all patient and control subjects. Results: A significant decrease in the level of serum and tears of total antioxidant status in diabetic cataract and cataract associated with glaucoma as compared to the control subjects. A highly significant increase in the level of nitric oxide, endothelin-1 in diabetic cataract and cataract associated with glaucoma but no significant changes in all parameters in senile and congenital cataract. Conclusion: A direct correlation exists between the level of total antioxidant status, nitric oxide, and endothelin-1 in serum and tears of diabetic cataract and cataract associated with glaucoma only, while in congenital and senile cataract a correlation does not exist.