Chapter 1. Introduction Cardiovascular disease is one of the leading causes of death globally. Every year, an estimated 17 million people die from heart disease globally. In Singapore, approximately 15 people die of heart disease every day and such deaths accounted for 32.4% of the total death rate in 2008. There are many forms of cardiovascular diseases, one of which is atherosclerosis. Cardiovascular mortality and morbidity are highly correlated with atherosclerotic disease. Atherosclerosis is a chronic process involving oxidized low-density lipoproteins (oxLDL) in the vessel wall. The body’s immune response towards damage is to release macrophages and T cells to engulf these ox-LDLs, forming foam cells. These cells eventually rupture thus releasing more oxidized cholesterol. The accumulations of substances such as cholesterol, fats, calcium and fibrin eventually build up into a plaque, causing the narrowing of the arteries, the loss of elasticity of the arterial walls, reduced blood flow and increased blood pressure1, 2. Gamma- glutamyltransferase (GGT) is known to be involved in the oxidation process of LDL and active GGT has been observed in atherosclerotic plaques3, 4. Prospective studies have also recently shown that plasma GGT levels can apparently predict the outcome of mortality in cardiovascular disease over time5. 1.1 Free Radicals A free radical refers to any species containing one or more unpaired electrons, each occupying an atomic or molecular orbital by itself (Halliwell & Gutteridge, 2007). The superscript dot behind the chemical name indicates that a species is a free radical (eg. O2). The presence of one or more unpaired electrons is able to cause free 1 radicals to be attracted towards a magnetic field. In general, free radicals are more reactive than non-radicals, even though the reactivity of radicals is widely variable6. There are free radicals present in vivo. However, most molecules in vivo do not exist as free radicals, which can be created from losing or gaining an electron from nonradicals7 among other mechanisms. Free radicals and other reactive species are continuously generated in vivo during physiological and pathological processes. 1.2 Reactive Species 1.2.1 Reactive Oxygen Species Reactive species is a collective term, which includes oxygen radicals and non-radical derivatives of oxygen such as H2O2 or hypochlorous acid (HOCl). The table 1.2.1.1 below shows nomenclature of reactive species of oxygen found in vivo (adapted from Halliwell)7. Reactive Oxygen Species Radicals Non-Radicals Superoxide, O2 Hydrogen peroxide, H2O2 Hydroperoxyl, HO2 Peroxynitrite, ONOO Hydroxyl, OH Peroxynitrous acid, ONOOH Peroxyl, RO2 Nitrosoperoxycarbonate, ONOOCO2 Alkoxyl, RO Hypochlorous acid, HOCl Carbonate, CO3 Hypobromous acid, HOBr Carbon Dioxide, CO2 Ozone, O3 Table 1.2.1.1 Nomenclature of ROS of both radicals and non-radicals. 2 Reactive oxygen species (ROS) such as H2O2, O2 and OH are generated in cells via several pathways8. Cellular energy is dependent on ATP production through electron transport reactions in which O2 accepts elections and H+ is reduced to water. The electron transport though the mitochondrial respiratory system is highly efficient (shown in figure 1.2.1.1). However, the leakage of an electron to O2 results in the production of O2. This leakage is believed to be mediated by complex I, coenzyme Q and ubiquinione and its complexes9. Therefore, mitochondria are thought to be a major site of ROS production in vivo10. Figure 1.2.1.1 shows the metabolic pathways of ROS formation. ROS such as H2O2, O2 and OH are generated in cells via several pathways. ROS can also be generated by ionizing radiation. O2- is generated from a number of pathways such as leakage of electrons from mitochondria, cytochrome P450 reductase, hypoxanthine/ xanthine oxidase, NADPH oxidase, lipooxygenase and cyclooxygenase. Superoxide dismutase 3 then converts O2 into H2O2, which is then degraded to H2O by glutathione peroxidase and catalase. Thioredoxin (TRX) has H2O2 reducing properties and refolding oxidized protein function. H2O2 produces highly reactive radical OH by the Fenton or the Haber – Weiss reactions (Figure is adapted from Kamata H and Hirata H)8. 1.2.2 Hydrogen Peroxide H2O2 is a pale blue covalent viscous liquid that boils at 150oC. Figure 1.2.2.1 (below) shows the structure of H2O2 in its gaseous and solid state (Adapted from www.commons.wikimedia.org). Figure 1.2.2.1. Structure of H2O2 in gaseous and solid state, varying in the degrees in angle between H-H atoms and H-O atoms. H2O2 plays an important role as an inter- and intra-cellular signaling molecule11, 12. As such, a basal level of H2O2 must be present in the cells. H2O2 is generated in almost all tissues and at significantly high rates in mitochondria by monoamine oxidase in the brain or by dismutating O2 from the electron transport chain via superoxide dismutase (SOD) in the equation below. (2 O2 + 2 H+ → H2O2 + O2) Cellular H2O2 and other peroxides are eliminated by seleno-enzyme SH peroxidase4 catalysed reduction, using glutathione (GSH) as substrate13, 14 . H2O2 reacts only moderately with other biological molecules, however it is unable to directly oxidize DNA, lipids and most proteins (except hyper- reactive thiol groups or methionine residues7). H2O2 is readily converted to OH, either by –ray or ultraviolet (UV) light radiation15. Thus H2O2 can be highly reactive in the presence of Fe and Cu ions, inducing oxidative damage on cells through Fenton reaction or Haber- Weiss reaction generating OH. The Haber-Weiss reaction generates OH from H2O2 and O216. It can occur in cells and may be due to oxidative stress. The first step in the catalytic cycle involves the reduction of ferric ion to ferrous: Fe3+ + O2  Fe2+ + O2 The Fenton reaction involves the formation of hydroxyl radicals (OH) from H2O2 in the presence of Fe2+ or Cu+ ions7: H2O2 + Fe2+  Fe (III) + OH- + OH H2O2 + Cu+  Cu2+ + OH- + OH SOD converts O2 into H2O2, which in turn is degraded to H2O by several cellular enzymes15 such as catalase and GSH peroxidase in the GSH pathway. H2O2 when present in low concentration, is able to enhance proliferation of certain cell types7. However, the concentration of H2O2 at about 10-100 µM is toxic to cells, causing either senescence or apoptosis in vitro. Some enzymes are known to generate H2O2. Ascorbate and certain flavonoids are compounds that are commonly known to generate H2O2 in cell culture media. This 5 H2O2 generation may cause cell culture artefacts resulting in misinterpretation of the results with effects on cells17. 1.3 Antioxidant Defence Enzymes 1.3.1 Superoxide Dismutases (SODs) Exposure to free radicals led organisms to evolve an antioxidant defence system. The discovery of SODs provided the basis for the current understanding of antioxidant defence since it led to the postulation of the superoxide theory of O2 toxicity and the realization that free radicals are important metabolic products18. There are a few forms of SODs such as CuZuSOD, MnSOD, FeSOD, an E. coli hybrid SOD enzyme containing subunits of manganese and iron in the same molecule18, cambialistic SOD19 and NiSOD20. Of all these SODs, the 2 most important SODs are CuZnSOD and MnSOD as they are present in animal tissues and play a more important role in antioxidant defence. CuZnSOD is a superoxide dismutase containing copper and zinc. SOD enzymes are highly efficient in removal of O2, however they can promote a few other reactions in vitro18, 21. An example is when nitroxyl anion (NO-) reduces Cu2+ in the presence of O2, CuZnSOD can catalyze conversion of NO- to NO22. NO- + O2 + 2H+  NO+ H2O2 Most CuZnSODs are rather resistant to heating, attack by proteinase and denaturation and are present in most eukaryotic cells. CuZnSOD is located in the cytosol in animal cells, however some appears to be present in lysosomes, nucleus and the space between inner and outer mitochondrial membranes18, 23. 6 Another form of SOD is known as Manganese SOD (MnSOD). It is also known as SOD2. It contains manganese in its active site as Mn (III) in the ‘resting’ enzyme. Unlike CuZnSOD, almost all MnSODs are more labile to denaturation by heat or organic solvent/ detergents7. MnSOD are widely present in bacteria, plants and animals. For most animals’ tissues, MnSODs are largely found in the mitochondria. The relative amount of MnSOD and CuZnSOD present in animals are tissue and species- specific18. 1.3.2 Glutathione Peroxidase family (GPx)24, 25 The glutathione peroxidases (GPx), selenium- dependent enzymes26 are capable of reducing H2O2 to H2O with the oxidation of GSH. H2O2 + 2GSH  GSSG + H2O Glutathione peroxidase was first discovered in animal tissues in 1957 and is less common in plants or bacteria24. The GPx enzymes can be found across different animal tissues (see table 1.3.2.1 below)7 and are widely specific for GSH as a hydrogen donor. GPx enzymes can be inhibited by mercaptosuccinate 27 and are able to act on peroxides other than H2O2, reducing them to alcohols24, 25. LOOH +2GSH  GSSG + H2O + LOH There are at least 4 types of GPxs 7, 25 , one of which is a ‘classical’ enzyme often known as cytosolic GPx or GPx1. GPx3 is a glycoprotein and low levels can be found in mammalian plasma. It can also be found in other extracellular fluids such as milk, seminal fluid, amniotic fluid and. it originates mainly from the kidney. Another type of GPx is found in cells lining the gastrointestinal tract. It is commonly known as the 7 intestinal GPx or GPx2. GPx2 functions to metabolise peroxides presence in ingested food lipids and those generated during lipid oxidation in the intestine. The human liver has been found to contain GPx2, GPx1 and GPx4. GPx4 is known as the phospholipid hydroperoxide glutathione peroxidase. It has the unique ability to reduce H2O2, synthetic organic peroxides, fatty acids and esterified cholesterol hydroperoxides. GPx4 can reduce peroxidized fatty acids within membranes and lipoproteins to alcohols. Table 1.3.2.1 shows the presence of glutathione and other enzymes in different organisms such as different parts of rat tissues, human tissues and different conditions of E.coli. The data is compiled from a wide range of publications7 and the studies show that there is low or no GPx levels present in spinach chloroplasts and bacteria and high levels of GPx in both rat and human liver. (Adapted from Free Radicals in Biology and Medicine, Barry Halliwell and John Gutteridge7) 1.4 Oxidative Stress 8 Cellular thiol redox state is a crucial mediator for multiple metabolic, signalling, and transcriptional processes in cells, balancing between normal oxidising and reducing conditions, which is essential for the normal function and survival of cells28. However when there is an alteration in the redox homeostasis, due to the excessive production of ROS/RNS (i.e increased exposure of O2 or excessive activation of systems producing RS)7 or that there is an impairment of antioxidant defence system (due to mutations affecting endogenous antioxidant defences or diminished essential dietary constituents)28, oxidative stress occurs (Figure 1.4.1). Figure 1.4.1 shows the imbalance of ROS/RNS and antioxidants, disrupting the redox homeostasis and eventually resulting in oxidative stress. (Adapted from Dalle-Donne et al, 200828). The term oxidative stress was first coined from the title of the book – Oxidative Stress, in 198529 and later defined as a disturbance in the pro-oxidant- antioxidant balance in favour of the former30, which leads to oxidative damage. The consequence of oxidative stress is dependent on cell types and severity of oxidative stress level (as 9 shown in figure 1.4.2 below), which may result in increased proliferation, adaptation, cell injury, senescence or cell death7. Figure 1.4.2 showing how cells respond to oxidative stress. (Adapted from Halliwell, 2000.31) The diagram shows that resting cells are usually in a reduced state and as the oxidative stress increases, so does the damage to the cells to the point of severe mitochondrial damage, excessive DNA damage and apoptosis or other modes of cell death. 1.5 Glutathione Glutathione is almost ubiquitous in cells32 and plays a key role in reduction processes by maintaining thiol groups of intracellular proteins26. It is also known to be involved in many metabolic processes such as maintaining communications betweens cells though gap junctions33, preventing protein-SH groups from oxidising and crosslinking. Although these functions are associated with protection against RS, maintenance of a suitable thiol redox balance with low- molecular- weight and protein thiols are also crucial for cellular homeostasis32. 10 GSH is a tripeptide with sequence -Glutamyl-Cysteine-Glycine as shown in figure 1.5.1 below. The oxidation of the thiol group from the cysteine moieties of 2 GSH molecules, results in the formation a of disulfide bond to form GSSG26. Because of the structure of GSH and GSSG, both of them are water-soluable26. Figure 1.5.1. Structure of Glutathione, taken from Free Radicals in Biology and Medicine, Barry Halliwell and John Gutteridge7. Glutathione present in mammalian tissues is mostly in the reduced form; the ratio of reduced to oxidized GSH in normal cells is very high (100:1)7. 1.5.1 Function of GSH Glutathione has two major functions; (1) as a substrate for GPx- mediated reduction of oxygen free radicals and (2) for biotransformation of exogenous compounds catalyzed by glutathione-S-transferases26. Glutathione also plays an important role in 11 protecting against ionizing radiation34, assists in supplying copper to CuZnSOD35 and acts as a cofactor for enzymes in different metabolic pathways36, 37. Glutathione also plays a part in protein folding as well as degradation with the cleavage of disulphide bonds, of which insulin is an example38. Glutathione is known to behave as an antioxidant by scavenging RS intracellularly, reacting to generate thiyl (GS) groups and O239, 40. The O2 generated could be removed by SOD and the GSSG can be reconverted into GSH by glutathione reductase catalysing the reaction: GSSG + NADPH + H+  2GSH + NADP+. The reduction potential of GSSG to GSH, however varies from cytoplasm to endoplasmic reticulum to mitochondria due to concentration and to pH41. Glutathione’s reaction with ONOO can somehow convert ONOO to NO42. Glutathione also chelates copper ions and diminishes the generation of OH from H2O243. The need for GSH increases as oxidative stress increases. Therefore, it is important to maintain an adequate level of intracellular glutathione in the reduced (GSH) state under normal conditions in vivo. 1.5.2 Glutathione synthesis and degradation Glutathione is synthesized in the cytoplasm of all cells with its highest production and disposition in the liver cells7, 26. It is known that GSH is secreted into the plasma by the liver and for GSH synthesis in other tissues via different transporters44. Mitochondria, containing 10- 20% of the total cellular GSH, also require transporters in the inner membrane to bring GSH from the cytoplasm into the matrix45. Glutamate-cysteine ligase (also known as -glutamylcysteine synthetase, GCS) is the first step in GSH synthesis and the dipeptide product is converted to GSH by glutathione synthetase as shown below (Adapted from Dalton et al)46. 12 L-glutamate + L- cysteine + ATP  L--glutamyl-L-cysteine + ADP + Pi L--glutamyl-L-cysteine + ADP +Pi +glycine +ATP  GSH + ADP + Pi Cells can make the necessary cysteine from methionine or take it up from the surrounding fluids as cysteine and/ or cystine (disulfide form), which will then be reduced to cysteine in the cell7. Different transporters are used for cysteine and cystine. However both cystine and glutamate share the same transporter. Therefore, high levels of extracellular glutamate can decrease the synthesis of GSH in some cell types due to the slowing down of cystine entering the cells47. Mammalian cells however are unable to take up GSH as a whole and it therefore needs to be broken down into a glutamyl moiety, cysteine and glycine. The first step requires an enzyme called -glutamyltranspeptidase (or -glutamyltransferase or GGT), which is located on the plasma membrane with its active site facing outwards and acting on extracellular GSH. This is done by removing the -glutamyl moiety either by hydrolysis or transferring to an acceptor7. The cysteine- glycine dipeptide is hydrolysed by dipeptidase to give cysteine and glycine for uptake into the cell. The glutamylamino acid is also taken up and converted to 5- oxoproline, which is then converted to glutamate (glutamic acid). In the case of glutamyl-cysteine, it can be directly converted to GSH48. The figure 1.5.2.1 below shows the synthesis and degradation of the glutathione cycle. 13 Figure 1.5.2.1 shows biosynthesis and degradation of the glutathione cycle. (a) Glutamylcysteine synthetase; (b) glutathione synthetase; (c) -glutamyl cyclotransferase; (d) oxoprolinase; (e) -glutamyltranspeptidase; (f) dipeptidases (including leucine aminopeptidase). This diagram is adapted from late Professor Alton Meister and the American Society of Biochemistry and Molecular Biology. 1.5.3 Redox Signaling Thiol groups are essential for protein function and total level of protein-SH in cells. Protein-S-glutathionylation is defined as the formation of mixed disulfides between an exposed protein thiol and glutathione. This process occurs in response to cells undergoing oxidative stress26. Therefore the ability to reverse this process is important to cells in response to oxidative damage and may have a role in redox signalling. Reverse glutathionylation or deglutathionylation can be achieved by glutaredoxin 14 proteins (GRx). Glutaredoxin, also known as thioltransferase, was originally discovered as a cofactor of ribonucleotide reductase in E. coli but was later found to be present in many organisms and animals49, 50. Glutaredoxin can be found in both mitochondria and cytosol and can be reduced directly by GSH. It is involved in catalysing thiol-disulphide interchange and the repair of glutathionylated proteins. There are 2 types of mammalian GRx; GRx1 is located in the cytoplasm of the cells, while GRx2 is found to be analogous to the glutathione recognition site of GRx1 and is directed to the mitochondria and nucleus. Both GRx1 and GRx2 are important for modulating the S-glutathionylation status and corresponding activities not just for mitochondrial and nuclear proteins, but also for certain nuclear transcription factors that translocate from cytosol to nucleus49. 1.5.4 Effects of deficiencies in GSH metabolic enzymes Glutathione metabolism plays an important part in the development of humans and animals. Deficiencies in GSH metabolic enzymes can lead to oxidative stress, which in turn plays a key role in aging and pathogenesis conditions such as seizure, Alzheimer's disease, Parkinson's disease, liver disease, sickle cell anemia, HIV, AIDS, cancer, heart attack, stroke, and diabetes47. Patients with glutathione reductase deficiency exhibit cataract and premature aging similarly to mice treated with buthionine sulphoximine, an inhibitor of glutamylcysteine synthetase47. Knockout of -GCS in mice has proven to be embryonic lethal46. Mice lacking GGT showed increased plasma and urinary GSH together with signs of ageing and myocardial infarction, which signifies this enzyme’s great importance in the maintenance of tissue GSH levels in mice46. Human lacking GGT, on the other hand, showed normal 15 cellular GSH levels, elevated GSH plasma levels and few symptoms as far as they have been studied47. 1.6 Quinones Quinones is a general term for a ubiquitious class of compounds which are common in several natural products and endogenous biochemicals or generated through metabolism of hydroquinones and/ or catechols51. Some quinones are potent redox active compounds, whereby they can undergo enzymatic (i.e., P450/P450 reductase) and non- enzymatic redox cycling with their corresponding semi- quinones radicals, resulting in superoxide anion radical generation52-54. Quinones and semiquinones are able to generate OH from H2O2 by enzymatic or spontaneous dismutation of superoxide anion radicals, involving iron ions or other transition metals51. Quinones are also Michael acceptors such that damage due to these species sometimes results from covalent binding with cellular nucleophiles. For example, many quinones can react readily with sulfur nucleophiles, such as GSH or cysteine residues on proteins, leading to depletion of cellular GSH levels and/ or protein alkylation51. Quinones exist naturally and can be found in cigarette smoke and widely in nature55. Semiquinones undergo redox inter- conversion to quinones and hydroquinones to produce O2 and H2O2. Quinones can be used as predator repellents56, 57, in cancer chemotherapy58 and as antibiotics59, 60 . Superoxide can exist in equilibrium with quinones and semiquinones (shown below). Semiquinone + O2  Quinone + O2 16 Quinones can play roles both as scavengers and generators of O2, which is dependent on the position of equilibrium and pH, since degree of protonation affects their reduction potential51, 61 . Dismutation of O2 in aqueous solution favors the synthesis of semiquinone and O2 by removing O248. Quinones also have a role in complex III of the electron transport chain in the mitochondria, as the hydrophobic coenzyme Q (ubiquinone or CoQ). The quinone ring of the coenzyme Q can be reduced to a quinol in a 2e reaction62: Q + 2e +QH2 The operation of the Q cycle in complex III results in the reduction of cytochrome c, oxidation of ubiquinol to ubiquinone, and the transfer of four protons into the intermembrane space63. Ubiquinol (QH2) binds to the Qo site of the complex III via hydrogen bonding to His182 of the Rieske iron-sulfur protein and Glu272 of cytochrome b. Ubiquinone (Q), in turn, binds the Qi site of complex III. Ubiquinol is divergently oxidized to the Rieske iron-sulfur protein and to the bL heme. This oxidation reaction produces a transient semiquinone before complete oxidation to ubiquinone, which then leaves the Qo site of complex III64, 65. 1.6.1 Mechanism of quinone toxicity Quinones can be toxic to cells via two mechanisms- reaction with glutathione (GSH) and redox cycling51. Quinones and semiquinones may react with GSH and –SH groups on proteins. The reaction with GSH may be non-enzymatic and/ or can be catalysed by glutathione- S- transferase (GST) enzymes, resulting in GSH depletion in the cell. This GSH depletion may result in renal failure due to conjugates of hydroquinones interacting with GSH, thus causing oxidative stress51. Redox cycling refers to a compound continuously being reduced and reoxidized by O2 to generate 17 O2. Most of the O2 will be converted to H2O2, which will be dealt by the glutathione cycle7. 1.6.2 Menadione Menadione is a synthetic compound lacking the isoprenoid side- chain on the vitamin K (a quinone). However, it still exhibits vitamin K activity in animal tests and is thus named as vitamin K37. The structure of menadione is shown below in figure 1.6.2.166. Menadione is able to cause haemolysis in glucose-6- phosphate dehydrogenase deficiency by oxidising and precipitating oxyhaemoglobin protein, forming semiquinone67. Fischer-Nielsen and colleagues68 showed that a high concentration of menadione is able to deplete GSH and increase the formation of O2 and H2O2 in rat hepatocytes. This also resulted in cell membrane blebbing and increased intracellular free Ca2+ due to oxidative stress69. Menadione is able to cause DNA damage indirectly via Ca2+- dependent nucleases68. Figure 1.6.2.1. Structure of menadione, taken from Helmenstine A.M66. 1.7 Gamma glutamyltransferase 1.7.1 Gene 18 Gamma glutamyltransferase (E.C.3.2.2.2) is an enzyme that can be found widely in bacteria70, 71 , plants72, 73 and the animal kingdom ranging from nematodes74 to humans. In humans, the GGT genes are located on chromosome 22q1175, 76. There are seven or more GGT genes present in humans77, 78, however only one gives rise to complete and functional protein. Although rodent and human GGT genes may show similarities in many ways such as exonic structures and existence of multiple forms of mRNA79, rodent GGT genes appear to be single-copy genes that have different sequences from the human genes80. GGT genes have many promoters and gene transcripts giving rise to the same protein. However, this is thought to be tissuespecific and also occurs during different phases of development77, 78. The difference in genetic expression is closely related to the metabolism of GSH81. 1.7.2 Protein The translation regulation by the 5’ untranslated region of a GGT mRNA, was initially found using HepG2 cells, and appears to serve as a tissue- specific translational enhancer82. Gamma- glutamyltransferase gene is translated into a single precursor protein that is catalytically inactive83, which undergoes an autocatalytic process into a heavy and light chain84. The heavy chain, which has the aminoterminal sequence, has a single intracellular transmembrane domain that anchors itself to the cell membrane. The light chain, which contains the active site, is situated in the extracellular domain. The heavy chain not only holds the light chain to the cell membrane, but also modifies its catalytic enzymatic activity84. There are as many as eight potential sites for glycosylation and the proteins in different tissues or animals are heavily glycosylated with variable heterogeneity84. HepG2 cells, on the other hand, have been reported to express a single active polypeptide form of GGT85, 86. In 19 normal human tissues, Hanigan and Frierson87 showed using western blot and immunohistochemistry that most of the immunopositive cells were epithelial cells, such as the epithelial lining of the ileum, gallbladder and epididymis, seminal vesicle and prostate. Positive staining of GGT could be detected in the bile ducts and canaliculi of liver and intense staining was seen in proximal tubule kidney cells81, 87. Stromal cells in bladder and colon were stained focally for GGT87. In human foetus, positive GGT staining was demonstrated in kidney proximal tubules, intestinal epithelium, bile ducts and canaliculi, pancreatic ducts and cortical epithelium of the adrenal cortex87. Macrophages in many tissues were also shown to be immunoreactive for GGT protein87. However, fat and muscles showed consistent negative results for GGT staining87-89. Fibrous stroma was mostly negative for GGT as well. Traces of positively stained fibroblasts however could be seen in certain sections of bladder, colon, liver, breast and ovary87. 1.7.3 Enzymatic Activity The enzymatic activities of GGT vary greatly between different normal human tissues and among different stages of embryonic development. The activities also vary between normal tissues to neoplastic tissues and normal cells to transformed cells in vitro87. An example is demonstrated by Hanigan and Frierson87 where the GGT enzymatic activity was measured to be approximately 103 Units/g protein in kidney homogenates and less than 1 Unit/g protein in normal human myometrium. Normally in the kidney, GGT acts to cleave extracellular GSH, eventually forming 3 amino acids to be reabsorbed from the glomerular filtrate87. Previous studies have shown that inhibition of GGT in the kidney causes glutathionuria, resulting in the loss of glutathione from the body90, 91. Studies with GGT- deficient mice have also shown 20 that kidney GGT plays an important role in the recovery of cysteine from urinary glutathione and the lack of it cause extensive excretion of cysteine in the urine92, 93. The GGT activity present in human liver homogenates is about one-fifth of that in the human kidney94. The GGT activity in the kidney shows that the enzyme level is initially low in neonates and gradually increases as the neonates grow and reaches a maximum at maturity95, 96 . There are other cell types in humans that contain significant GGT activity. An example is the astrocytes found around the blood vessels in the brain97, conjugating 98 . They are believed to play a role in the blood-brain barrier by xenobiotics or metabolizing vasoactive leukotrienes. Gamma glutamyltransferase can be also found in human white blood cells with its activity varying with cell type and stages of differentiation99-103. Even though the exact molecular mechanism of GGT action is not thoroughly understood, it is known that GGT transfers the glutamyl moiety to another amino acid, an acceptor. The reaction of GGT appears in this general form: Gamma- glutamyl-X + acceptor  Gamma- glutamyl-acceptor + X A wide range of compounds can be used as a gamma-glutamyl donor or as acceptor. The most natural substrate is GSH (gamma-glutamyl cysteinyl glycine) and the most active and common acceptor substrate is glycylgycine94. Artificial substrates such as gamma- glutamyl-p-nitroanilide and gamma- glutamyl-3-carboxy-4-nitroanilide were developed for the measurements of GGT activity. GGT activity is usually measured by a kinetic spectrophotometric method developed by Szasz104. GGT has been shown to be a relatively stable enzyme in vitro105. According to his method104, the ideal measurement of GGT kinetic activity with human serum or plasma is to be done at a 21 constant 37oC environment. However, the measurement of GGT activity is often also done at different temperatures such as 25oC and 30oC. In order to correct for the change in activity, a calculation has been formulated to convert activity measured at 25oC to that at 37oC by multiplying by 1.78 and of 30oC at 37oC by multiplying by 1.31106. 1.8 Normal Function The activity of GGT was initially observed to be greatest in tissues with transport function such as kidneys and in the bilary system94. As previously mentioned, GGT plays an important role in transportation of amino acids, though a sequence of reactions forming the “gamma-glutamyl cycle”. GGT is important for the availability of the amino acid cysteine that is usually undersupplied intracellularly. Extracellular glutathione is broken down at the cell membrane to its constituent amino acids such that they can be readily taken up by GGT- possessing cells for use to synthesize intracellular glutathione94. De-Oliveira and colleagues107 have shown that rats that were fed with low sulphur- containing amino acid diets (rice and bean diet), have lower glutathione and higher GGT levels as compared to control rats. When methionine was added to the low-sulphur diet, the glutathione level increased and GGT value fell to normal. This reciprocal relationship between intracellular GSH and GGT is assumed to relate to the production of cysteine from circulating GSH107. 1.9 Cellular Pathology The distribution and concentration of GGT present in tumours show a number of differences when compared to normal tissues94. Hanigan et al. have compared the GGT expression of carcinogenic and normal tissues and have found that most of the 22 carcinoma cells from GGT- positive organs were themselves positive. Also, carcinomas of lung and ovary showed GGT positive staining whereas normal bronchial and ovarian epithelial tissues showed no staining for GGT108. Hanigan et al. have also reported that a decreasing amount of immunoactivity of GGT is observed from normal breast tissues to benign and malignant tissues progressively109. However, another study with ovarian cells showed opposite effect, i.e. increasing amount of GGT immunoactivity changing from normal tissues – benign- malignant cells progressively110. It seems that there is no uniform behavior across all human cancer types over the expression of GGT94. Hanigan et al. 108 have also found that human hepatocellular carcinomas (HCC) showed GGT activity. Rat hepatomas were found to be GGT positive whereas mouse hepatomas showed negative. This could be due to the lack of GGT promotor sequence in mouse111. This suggests that GGT expression is not a universal feature across species94. 1.10 Serum GGT The activity of GGT in blood provides a sensitive indication for liver tissue alteration, which may suggest pathological disorders. Schiele et al.112 observed that the reference range for serum or plasma GGT (Figure 1.10.1) shows significant differences between genders with age. This result is consistent with other findings with confounding factors of gender113-116 and age112, factors such as pregnancy121, 122 117-120 . There are a series of other confounding , childbirth123, race115, smoking115, the use of oral contraceptives112, 115 and exercise124, which affect the levels of serum GGT. 23 Figure 1.10.1 shows the mean of serum GGT activity with different age group and gender. The results showed that serum GGT started about the same range in children but started to diverge significantly by gender at age 16- 20. This graph was taken from Schiele et al112. 1.10.1 Serum GGT in liver disease Serum GGT has been used clinically for decades as a biomarker for liver function. It was first adopted by Szczeklik et al.125 who showed the changes in serum GGT in different types of liver diseases over time and compared GGT with other enzymatic markers. GGT measurement is a simple and sensitive assay detecting abnormal values in liver disease patients regardless of causes, and high values in cholestasis94. Trends 24 in serum GGT values were also used to detect the rejection of liver transplants 126. However, the drawback of this test is that it is not able to distinguish specific diseases or conditions such as excessive alcohol intake or diabetes, which result in high serum GGT94. 1.10.2 Serum GGT in liver cancer High level of GGT was found in non-alcoholic steatohepatitis (NASH), which is a hepatic disease associated with diabetics and obesity, without the alcohol127-129. Clinical studies have also shown a high prevalence of abnormal serum GGT in patients with primary or secondary liver cancer. A number of experimental studies have been performed to understand if the GGT isoforms have any relationship with liver cancer, especially human hepatoma (HCC). There were a few promising results, but nothing conclusive due to the variety of GGT isoforms separated using different techniques, and the lack of standard nomenclature94. The general deduction from the above mentioned publications suggested that the GGT isoform that was not associated with lipoproteins and had greater electrophoretic mobility, was mostly likely to be associated with HCC94. An investigation comparing the different GGT isoforms in human HCC against GGT in normal or cirrhotic liver showed reduced electrophoretic mobility130. However, the mobility rates varied between samples, patients and isoeletric points of GGT isoforms in HCC against control94. Therefore, further work has to be done on improving and standardizing the measurements of GGT isoforms. 25 1.10.3 Serum GGT and alcohol intake Serum GGT was first used as a liver function test in the early 70s, in which GGT was particularly sensitive to alcoholic liver disease131. It was later discovered that serum GGT was elevated in a high proportion of alcoholics even when there was no sign of liver disease132-134. The observation that GGT is sensitive to alcoholic liver disease, could be used for detection of potentially harmful drinking and reduce the potential harm caused by alcohol94. A single dose of alcohol was shown to have no effect on serum GGT135-137, but consumption of 63g per day for 5 weeks produce a significant increase in GGT138. Health screening was done on both men and women by Nagaya et al.139 to understand the correlation between alcohol intake and serum GGT distribution. The results from serum samples showed that there was a greater proportionate increase in GGT than in aspartate transaminase (AST) or alanine aminotransferase (ALT) as shown in figure 1.10.3.1 below. 26 Figure 1.10.3.1. The graph was taken from Nagaya et al.139 and the result from serum samples showed the relationship between alcohol intake and average value of GGT, AST and ALT in middle- aged Japanese men. The error bars refer to 95% confidence interval. The increase in GGT enzyme activity is much greater than that of AST and ALT. 1.10.4 Mechanism of GGT increase in liver disease The mechanism of the increase in serum GGT in liver diseases is poorly understood. However Teschke et al.140 found that normal human serum and hepatic GGT is 20fold higher than normal rats. Satoh et al. have also found that higher values of GGT 27 were seen in patients with alcoholic or drug-induced hepatitis compared to viral hepatitis141. Serum GGT of the alcoholic or drug- induced hepatitis patients was found to be approximately six- fold higher compared to normal serum GGT and liver GGT was found to be about three-fold higher than normal. Two conclusions could be deduced from these results94. Firstly, hepatic GGT is increased in some types of liver disease. Since the increases in serum GGT and hepatic GGT are not similar, the increase in serum GGT is not simply release of enzyme from damaged cells and it could be that the enzyme expressed on cell surface is able to release into the circulation more readily than usual. It was also suggested that the release of GGT into serum could be due to bile acids acting on the cell membrane. Secondly, the increase in GGT is somehow associated with, or specific to some extent for, alcohol-related disease. The near- normal level of hepatic GGT in some liver diseases could result from loss of newly synthesised GGT from the cell surface into the circulation. 1.10.5 Serum GGT and cardiovascular disease As mentioned previously, serum GGT has commonly been used as a biomarker for liver function test or alcohol related disease. Recently, it appears that the relationship with cardiovascular disease (CVD) should be considered as well. There are many identified risk factors such as obesity142-145, blood pressure146, lipids and lipoproteins147, diabetes148, 149, exercise142, 150, 151, smoking152, 153, iron overload154 and coffee consumption155-158 that are associated with CVD and have been shown to increase serum GGT levels. The first report was in 1974 by Kristenson et al.159 and they showed that subjects with higher GGT had more incidences of sickness benefits over the next 21 years. In the same year, a mortality report showed that increasing death rates have an association 28 with the increasing level of GGT160. Later clinical papers by the same group, with more subjects and longer follow-ups, confirmed that GGT activity in serum could be a strong predictor of death caused by alcohol- related illness as well as myocardial infarction161. Conigrave et al.162 also showed, even with fewer subjects and shorter follow-up, with significance that GGT was able to predict mortality from liver disease among men, but not women. Recently, Hozawa et al.163 showed a strong positive shared relationship between serum GGT and CVD mortality in Japanese women with little to no alcohol consumption. Paolicchi and colleagues3 demonstrated though immunostaining that GGT is present and active in human atherosclerotic plaques. From their findings, they also suggested that the appearance of GGT in atherosclerotic plaques may derive from serum enzyme associated with circulating lipoprotein fractions4. The conclusion from these studies was that GGT is an independent predictor of mortality, and not merely a marker for liver disease or excessive alcohol intake. 1.10.6 GGT and Oxidative Stress Recent findings suggest that GGT levels could be a biomarker for oxidative stress as well. Duk Hee et al.5 found from his epidemiological studies with Coronary Artery Risk Development in Young Adults (CARDIA) that serum GGT even in the “normal” range might be an early and sensitive enzyme related to oxidative stress. This theory was supported with some experimental evidence. The serum antioxidant concentrations were shown to be inversely related to serum GGT level within its normal range in a dose dependent manner164. This inverse association between serum antioxidants and GGT was also confirmed with findings in the US populations by the third National Health and Nutrition Examination Survey 165 . Dietary factors such as 29 high intake of fruits are also inversely related to serum GGT in dose- dependent manner166. Another study demonstrated that other than alcohol consumption, meat intake can also positively predict future serum GGT level in a dose dependent manner166. This could be due to the haem and iron present in the meat causing oxidative stress; both haem and free iron from it can be critical catalysts in generating oxidative stress167-169. Based on the studies mentioned above, the increase in serum GGT could perhaps reflect a defence mechanism, i.e. induction of cellular GGT under oxidative stress165. Oxidative stress was introduced in vitro using menadione by Kugelman et al.170 on rat lung alveolar cells. Exposure of cells to menadione resulted in a dose dependent cytotoxic effect, with an initial decrease in intracellular glutathione and an increase in GGT mRNA and protein. Another study was done with high oxidative stress using hypoxanthine and xanthine oxidase on rat epididymis. The results show that gene expression of GGT mRNAs II-IV segments of rats was up-regulated up to 70% upon oxidative stress in a dose- dependent manner171. 1.11 Relationship between cellular and serum GGT The mechanism and relationship between cellular GGT and serum GGT has always been of interest. As previously suggested by Whitefield94, the differential increase in serum and cellular GGT precludes simple release from damaged cells. Recently, Franzini et al.172 compared the soluble GGT secreted by cultured melanoma, prostate cancer and bronchial epithelial cell lines with plasma GGT. He found that the GGT secreted by these cells is similar to a specific GGT fraction (b-GGT) found in the plasma. Since the GGT released by these cells contributes to only a subset of the GGT population present in the serum, this would probably explain why there is a higher 30 level of GGT present in the circulation compared to liver GGT and that there is an increase in serum GGT levels in non-liver related diseases. 1.12 Hypothesis. Elevation of serum GGT activity positivity predicts disease outcome in healthy volunteers and patients, one example being ischemic heart disease or other cardiovascular disease. Why is this so? Do cells under oxidative stress make and/or release more GGT into the blood? The objective of the work in this thesis is to further understand the links between cellular GGT and extracellular GGT, as well as to know whether there would be an increase in GGT level or increased GGT secretion upon oxidative stress. It was hoped thereby to explain why GGT seems to be a biomarker of oxidative stress. 31 2. Methods 2.1 Cell culture Human fetal liver (HFL), human embryonic kidney (HEK) and human hepatoma (HepG2) cells were generous gifts from Professor Sit Kim Ping (Dept. of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore). Human renal proximal tubules epithelial primary cells ( hPTE cells) were obtained from Lonza (Singapore). hPTE cells were cultured with Renal Epithelial cell growth Medium (REGM) basal medium supplemented with 1% serum and growth factors (Lonza, Singapore), while the other cells were maintained in Dulbecco’s Modified Eagle’s Medium with 1% (v/v) penicillin, 10% (v/v) FBS (PAA, Singapore), 5% CO2 at 37oC. The cells were routinely passaged every 2-3 days. HFL cells were cultured with HepatoZYME-Serum- Free Medium from Invitrogen (Singapore) for all experiments. 2.2 Cell Counting Trypan blue stain (0.4% w/v in phosphate buffered-solution) and the hemocytometer were used to determine total cell counts and viable cell number to facilitate cell seeding. The principle of staining is based on the ability of live cells to exclude dye staining whereas dead cells will be stained. Briefly, a cell suspension (10 µl) was mixed with 70 µl of trypan blue solution, and then a small volume (10 µl) of trypan blue-cell suspension mixture was transferred to each chamber of the hemocytometer with a cover- slip in place. Live cells without the dye were counted in the 4 big squares at the corners (25 grids in each squares). The number of cells per millilitre = average count per square multiplied by 104 (each square contains 0.0001 millilitre) and multiplied by dilution factor (8). 32 2.3 Assessment of Cell Viability 2-Methyl-1, 4-naphthoquinone (Menadione) was prepared in DMSO to give a 100µM stock. I then prepared a range of concentration- (10, 20, 30, 40, 50 µM) by diluting with HepatoZYME medium for the various timepoints. 30% stock hydrogen peroxide (H2O2) was diluted 1:100 in sterile H2O to give approximately 10mM concentration. The actual concentration was measured using spectrophotometry at absorbance 240nm wavelength at a molar extinction coefficient of 43M-1cm-1 (Long et al., 1999a). Various H2O2 concentrations (100, 200, 300, 400, 500 µM) were further produced by dilution with HepatoZYME medium for the various timepoint treatments. These concentrations were used to study the concentration- dependent cytotoxicity of menadione and H2O2 on human fetal liver cells and human renal proximal tubules epithelial cells. Renal epithelial cell growth medium containing growth factors and serum was use to dilute menadione and H2O2 concentration for renal proximal tubule epithelial cells. 2.4 MTS Assay Cell viability was measured by a colorimetric method using MTS [3-(4, 5dimethylthiazol- 2- yl) - 5- (3- carboxymethoxyphenyl)- 2- (4- sulfophenyl)] (Promega). The MTS solution is reduced by cells to formazan, which is soluble in tissue culture medium (Promega- CellTiter96 MTS assay manual). The absorbance of formazan was measured at 490nm. This assay measures the dehydrogenase enzyme activity found in metabolically active cells and procedures of this assay were carried out according to the Promega protocol. Cells in DMEM were seeded overnight at a density of 0.4 x 104 cells per well in 24-well plates. After exposure to menadione or 33 H2O2 dissolved in fresh hepatoZYMES for various time points, the supernatant was removed from each well and replaced with MTS solution (100µL) dissolved in 500µL of Earle’s Balanced Salt Solution (EBSS) per well. Cells were incubated at 37oC in the dark for 30 min. Absorbance at 490 nm was then determined using a microplate reader (InfiniteM200, Tecan) after shaking in the dark for 5 secs. 2.5 Western Blot Analysis- Cell culture Medium HFL cells were seeded in T75cm2 flasks with 15 x 105 cells per flask and treated with H2O2 and menadione at various time points. After treatment, serum- free cell culture media were collected and concentrated using a 15mL centrifuge tube with 10kDa membrane pore size protein concentrator (Millipore). These media were then centrifuged at 15,000rpm for 20 min at 4oC in a microfuge. The protein concentration from the cell culture media was determined with the Protein Assay Dye Reagent Concentrate (BioRad) and 50µg protein was boiled for 5 min at 95oC, together with protein dye before loading in a 10% (v/v) SDS-PAGE gel. Samples were run at 70 volts for 1.5 hours. The proteins on the nitrocellulose membrane were then probed with antibodies against GGT light- chain followed by the rabbit anti-goat secondary antibodies (Thermo-scientific). Detection was by enhanced chemiluminescence (Femto- Pierce). GGT horse enzyme (Sigma) was used as a standard protein control. RPTECs’ supernatants were also analyzed similarly. 2.6 Western Blot Analysis- Lysate Adherent HFL cells were washed once with ice cold PBS and trypsinized with 1ml of 0.25% trypsin-EDTA (Gibco-Invitrogen). Cells were incubated for 5 min and neutralized with serum- containing DMEM. Cell suspensions were divided into 2 tubes for cell lysis 34 and RNA extraction respectively. Both tubes were spun in the benchtop centrifuge machine at 1200 rpm for 5 min and their cell culture media were removed. 200µL of Radio-Immunoprecipation Assay (RIPA) buffer (Sigma-Aldrich), which consists of 50mM Tris-Cl, 150mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dedocyl sulfate, together with 5µl of proteinase inhibitors cocktail (Roche) was added to one of the tubes, vortexed and centrifuged at 13,000rpm for 15 min in a microfuge (Sorvall) to remove cell debris at 4oC. The cell lysate protein concentration was also determined with Protein Assay Dye Reagent Concentrate (Bio-Rad). Similarly, 50µg protein was boiled for 5 min at 95oC, together with protein dye before loading in a 10% (v/v) SDS-PAGE gel. The rest of the procedures were mentioned previously (western blot-cell culture medium), section 2.5. The proteins on the nitrocellulose membrane were then probed with antibodies against GGT customized heavy-chain antibody obtained from peptide sequences published by Hanigan and Frierson87, followed by the anti- rabbit secondary antibodies (Santa- Cruz Biotechnologies) and anti mouse beta-actin (Sigma) and anti mouse secondary antibodies (Santa- Cruz Biotechnologies). Detection was by enhanced chemiluminescence (Femto- Pierce). GGT horse enzyme (Sigma) was used as a standard protein control. RPTECs’ lysates were also analyzed similarly. 2.7 RNA Extraction Total RNA was extracted using 1ml of TRIzol (Invitrogen) added to the second tube of cells described in section 2.6. The cell pellet was well mixed until dissolved and incubated at room temperature for 5min. 300µl of chloroform was added to the TRIzol solution, mixed and incubated at room temperature for 3 min. This TRIzol-chloroform solution was then spun down in 1.5mL microcentrifuge tubes at 13,000 rpm at 4oC for 10 35 min in a microfuge. The aqueous phase of the mixture was added to a tube containing 0.45mL of 70% ethanol. The reagents were mixed well and incubated at room temperature for 10min. The remaining procedures were followed according to the RNeasy Extraction kit from Qiagen. 700µl of the RNA mixture were transferred to a RNA spin column (Qiagen) for pulse spin using a microfuge at 13,000rpm at 4oC for 5 sec. The eluant was discarded and this process was repeated with the remaining mixture. 700ul of RW1 reagent from Qiagen kit was added to the spin column and spun using a microfuge at 13,000rpm for 1 min before transferring to a new column tube (provided within the Qiagen kit). The RNA in the spin column was then washed twice with 500 µL of RPE (Qiagen) by spinning down at 13,000 rpm for 15 sec and then spun dry at 13,000 for 1min in a microfuge. The spin column was transferred to a 1.7 ml centrifuge tube and RNA was eluted twice with 20 µL of RNase-free water. RNA samples were stored at 80oC freezer. RPTE cells were analysed similarly. 2.8 Reverse Transcription The concentration of RNA was determined using a cuvette-free spectrophotometerNanodrop (Thermo scientific). 1ug of total RNA was mixed together with reverse transcription reaction buffer, 6 mM MgCl2, 200 µM deoxynucleotides, forward and reverse primers, reverse transcriptase and RNase- free water, to give a final volume of 20 µL per reaction tube. Thermocycler reaction steps were set in the following order: 25 oC for 10min, 37oC for 2 hour, 70oC for 10 min and lastly kept cool at 4oC in a PCR thermocycler machine (MJ Reseach-PTC100). cDNA samples were stored in a -20oC freezer. RNA samples from RPTE cells were also reverse transcribed by the same procedure. 36 2.9 Real- Time PCR Preparation and Analysis The cDNA was diluted in a 1:10 ratio with sterile milliQ water. A 9 µL volume of diluted cDNA sample was used together with 1 µL of Taqman human GGT probe (Applied Biosystems) and 10 µL of 2x Taqman mastermix (Applied Biosystems) per reaction. The samples were run using a real-time PCR machine (Applied Biosystems). The results were analyzed using real- time analysis and data were displayed as fold change. 2.10 Intracellular Glutathione Measurement GSH was measured according to Hissin and Hilf173 whereby o-phthaldialdehyde (OPT) is used as a fluorescent reagent. The reaction of GSH with OPT was carried out at pH 8.0, giving an optimal fluorescence when activated at 350nm and emission peak at 420nm. The fluorescence intensity decreases if pH falls below 8.0 and if the pH increases more than 8.0, the reaction will cause conversion of GSH to GSSG. Human foetal liver cells were seeded at a concentration of 2 x 105 cells/well into 6- well plates and treated with H2O2 and menadione at various concentrations and for different time points. The cells were washed twice with ice cold PBS. 100µl of ice cold 6.5% trichloroacetic acid (TCA) was added to each well and incubated for 10min on ice. 5µl of TCA extract was added to 96-well plates followed by the addition of 185 µL of KH2PO4KOH buffer (100 mM, pH 10.0), and 10 µL of freshly prepared o-phthaldialdehyde (10 mg/ml in methanol). Plates were incubated in the dark at room temperature (25oC) with gentle shaking for 25 min, followed by measurement using a Gemini Fluorescence plate reader at excitation 350 nm and emission 420 nm. Concentrations of GSH were determined from a freshly prepared standard curve of GSH (Figure 2.10.1 and figure 37 2.10.2). The intracellular glutathione in human RPTE cells was analyzed by similar procedures. The 2 standard curves showed very different readings, as they were done at different times and may be due to batch to batch variations. However, the RPTE cells or HFL cells were analyzed on the same 96- well plate as the standards for GSH and the results were analysed based on the resulting calibration curve. The standard curves were plotted with absorbance values against GSH concentrations. The last absorbance value for the 80µM GSH concentration was not included as it was an outlier and therefore the graph was plotted to give the best fit curve. Figure 2.10.1 shows the GSH standard curve for HFL cells. Concentrations ranging from 0- 80µM of GSH were used to determine and plot the curve. The calculations of intracellular GSH in HFL cells were based on the equation of this standard curve and a graph was plotted with the average absorbance values against the glutathione concentration. 38 Figure 2.10.2 shows the GSH standard curve for human RPTE cells. Concentrations ranging from 0- 40µM of GSH were used to determine and plot the curve. The calculations of intracellular GSH in human RPTE cells were based on the equation of this standard curve graph and was plotted with the average absorbance values against the glutathione concentration. Error bars were analysed based on 3 replicates. The last absorbance value with 80µM glutathione was excluded, as the value was an outlier. 2.11 GGT Enzymatic Activity Analysis GGT was first measured using a kinetic photometric method developed by Szasz104. GGT enzymatic activity was determined using L--glutamyl-p-nitroanilide as substrate and glycyl- glycine as transpeptidase acceptor174. This method was later modified by Franzini et al175. 150µl of substrate solution containing a final concentration of 10mM MgCl2 and 4.6mM gamma- glutamyl- p- nitroanilide (Sigma) dissolved in 0.1M of pH 7.5 Tris- HCl, were pre-incubated with 50µl of transpeptidase acceptor solution containing a final concentration of pH 7.5, 575mM glycyl- glycine dissolved in 10mL milliQ water, at 37oC for 10min. 100µl of serum sample or supernatant from cells were added into the 200µl of pre- incubated substrate-acceptor solution mixture in a 96-well plate. The amount of pnitroanilide present in samples was then analyzed using a kinetic spectraMAX190 plate reader, detecting at a wavelength 405nm wavelength for 20min at 1min intervals. The 39 activity of GGT (units/L) was calculated based on the averaged differences in absorbance reading per minute, multiplied by total volume divided by sample volume ratio, multiplied by 1 million divided by substrate molar extinction coefficient (which is 9200L.mol-1cm-1 for gamma- glutamyl- p- nitroanilide). 1 unit of GGT activity was defined as 1µmol of substrate transformed/ml/min. The GGT value was corrected for the protein concentration and finally expressed in units/mg protein. Human RPTE cells were analyzed similarly. 10 units of GGT horse enzyme (Sigma) were used as a standard. 2.12 Test for stability of GGT enzyme over time in patient serum. GGT enzymatic activity was analyzed similarly with human patients’ serum samples. Sera were kindly donated by volunteer patients involved in clinical trials in collaboration with Dr Raymond Seet of National University Hospital. The patients’ sera of each test group (normal and diseased) were analyzed to test their GGT enzymatic level. Samples were kept at -80oC. These sera were then thawed once a week at week1, 2, 3, 4 and 12 to measure the stability of the GGT enzymatic activity of serum. 40 Results 3. Cell lines and models of oxidative stress As previously mentioned, GGT is more in abundant in liver and kidney cells, and so experiments were carried out on human embryonic kidney cell lines (HEK293), human hepatocellular carcinoma cell lines (HepG2) and human Fetal Liver (HFL) cell lines. Human Renal Proximal Tubular Epithelial cell line ( hPTE cells), which is a primary cell line, was also used to carry out experiments. In order to induce oxidative stress, 2 models were used. The easiest and most direct way was to add H2O2 directly to cells and measure the amount of GGT gene expression, protein levels and enzyme activity. Another model used was the addition of menadione to cells. This was demonstrated by Kugelman et al.170 when he incubated rat lung alveolar cells with 50µM menadione for 15min before changing the supernatant to normal growth medium and incubation overnight. The mechanism of menadione action was reviewed in section 1.5.2 above. 41 3.1. Human Embryonic Kidney Cells (HEK 293) Cytotoxicity testing was done on HEK293 cells to determine the concentrations of H 2O2 and menadione that would affect cell viability no more than 20%. The limit of 20% cell death (or 80% viability) was selected to avoid confounding results due to excessive cell death. A range of menadione concentrations was tested and results are shown in Figure 3.1.1 below. Figure 3.1.1 shows the effects of varied concentrations of menadione added to HEK293 cells. The results are presented as mean  SE. Menadione concentration of 3,5 and 7µM was tested with replicates of n= 2 and menadione concentration from 10µM -30µM were tested with replicates of n= 5. As seen in figure 3.1.1, approximately 80% of cell viability was maintained with 10µM of menadione. 7µM and less of menadione concentration seemed to show slight (but not significant) cell proliferation, whereas 20µM and 30µM of menadione concentration showed at least 40% cell death in an increasing dose- dependent manner. Similar experiments were performed with a range of H2O2 concentrations as shown in Figure 3.1.2. 42 Figure 3.1.2 shows the effects of varied concentrations of H2O2 added to HEK293 cells. The results are presented as mean  SE. H2O2 concentrations of 40µM, 60µM 75µM, 150µM and 250µM were done with replicates of n= 2 and H2O2 concentration of 50µM, 100 µM, 200µM, 300- 500µM were done with replicates of n= 5. The results in figure 3.1.2 showed that the percentage of viable HEK 293 cells, after adding different concentrations of H2O2, was highly inconsistent even after 2-5 replicates, for unknown reasons. As a result of this inability to obtain consistent results, the use of the HEK293 cell line was discontinued for further experimental analysis. 43 3.2. Human Hepatocellular Carcinoma (HepG2) The MTS assay was performed on HepG2 cells with a variety of H2O2 and menadione concentrations. This assay was done after a 24h period. The graph in Figure 3.2.1 shows the percentage of viable HepG2 cells after treatment with a range of H2O2 concentrations. The experimental results demonstrated a good reproducibility and showed that there was an initial trend (but no significant increase) in cell viability with 10µM H2O2 concentration before going back to base level with 20µM H2O2 concentration. The percentage of cell death increased in a dose- dependent manner with increasing concentration of H2O2. This experiment also demonstrated that approximately 80% cell viability was maintained with 30µM H2O2. Figure 3.2.1 shows the effects of varied concentrations of H2O2added to HepG2 cells. The results are presented as mean  SE. H2O2 concentration of 1- 50µM was done with replicates of n= 3. Different concentrations of menadione were tested on HepG2 cells as shown in figure 3.2.2 below. A similar dose- dependent trend with menadione concentration was seen. There was a trend to a marginal increase in cell viability with 5µM menadione, which 44 later decreased with increasing concentration of menadione. There was no significant change with 5µM and 7µM of menadione concentration in term of cell viability, however 20µM and 30µM menadione concentrations showed significant viability decrease. Addition of 10µM menadione was shown to maintain an approximate 80- 90% cell viability. Figure 3.2.2 showed different concentration of menadione added to HepG2 cells for 24 h. The results were tabulated with mean  SE, where n = 3. However at this point I became aware of the work of Tate and Galbraith,85, 86 who reported that GGT the protein present in HepG2 cells was expressed as a single ‘active’ polypeptide form of GGT and did not undergo autocatalytic processing. Pawlak and colleagues176 also showed that alternate splicing at the transcriptional level, was found in HepG2 such that the truncated protein consist of mostly heavy chain. This resulted in lacking/ decreased catalytic activity in the extracellular medium of cells, since activity is situated in the light chain domain. Although both H2O2 and menadione gave reproducible results with HepG2 cells, the use 45 of HepG2 cells was also discontinued since GGT protein appears mutated in the HepG2 cell line. 46 4. Human Renal Proximal Tubules Epithelial Cells ( hPTE cells)- Primary cells 4.1 Cytotoxicity Assay (MTS) The MTS assay was also conducted in RPTE cells. Figure 4.1.1 below shows the action of different concentrations of H2O2 on RPTE cells over 6h, 9h and 24 h time points. Treatment of RPTE cells with H2O2 generally showed inconsistent effects with increasing concentration over time. The results suggested with (no significant difference) a slight decrease in cell viability by 6 h, followed by proliferation by 9h and a decline again by 24 h. Hydrogen peroxide did not seem to induce a drop in viability even after adding 500 µM of H2O2 for 24 h. Figure 4.1.1 shows the cytotoxicity assay of RPTE cells treated with different concentrations of H 2O2 over 6 h, 9h and 24h time point. The data are presented as mean  SE, where n= 4. Higher concentrations of H2O2 were tested to find out the concentration required to induce a 20% cell death. MTS assay was performed with higher concentration of H2O2 47 and results are shown in figure 4.1.2 below. The results from the 6 h and 9 h time point show a 40% decrease in cell viability with 700µM of H2O2 but surprisingly, an 80% cell viability with 1000 µM H2O2 before declining back down to about 40% cell viability with 1500 µM of H2O2. The 24 h time point, however, shows a 40% decrease in cell viability with treatment from 700 µM to 1500 µM H2O2. Based on the data shown below, 1000 µM of H2O2 appeared to be able to achieve a 20% cell death. Because of these variabilities, H2O2 was not used for further RPTE cell experiments. Figure 4.1.2 shows the cytotoxicity assay of RPTE cells treated with higher concentrations of H 2O2 over 6 h, 9 h and 24 h time point. The graph was plotted with mean  SE, where n= 3. 48 Similarly, figure 4.1.3 shows MTS assay results of RPTE cells treated with various concentration of menadione over time. The results show that 10µM of menadione is able to cause an approximate 20% cell death by 6 h and 9 h and about 90% cell death by 24h time point. 20µM of menadione shows a more drastic decrease in cell viability with 30% cell death by 6h, 40% cell death by 9h and 60% cell death by 24h. Acute addition of 50µM of menadione for 15 min, before changing to RPTE medium without serum was found to induce a 10% cell death by 6h and 9h and a 20% cell death by 24h. Figure 4.1.3 shows the cytotoxicity assay of RPTE cells treated with different concentrations of menadione over 6 h, 9 h and 24 h time points. Data are mean  SE, where n= 4. 49 Experiment were also done with lower concentrations of menadione. The results in figure 4.1.4 show that concentrations of menadione ranging from 5–9 µM did not cause any significant change in viability across the concentrations as well as time points. Figure 4.1.4 shows the cytotoxicity assay of RPTE cells treated with different concentrations of menadione over 6 h, 9 h and 24 h time point. Data are mean  SE, where n= 3, none of the changes is significant. Since the results shown in figure 4.1.4 did not show any significant difference among the different concentrations of menadione, we thus decided to use 10 µM menadione, or acute addition of 50 µM of menadione for 15 min, in further experiments with RPTE cells. 50 4.2 GSH Assay Glutathione was also measured in RPTE cells over a series of time points to understand the effect of oxidative stress. Data are shown in Figure 4.2.1. 10 µM of menadione (50 µM of menadione exposure for 15 min) were used to examine the effects on intracellular GSH levels at 6 h, 9h, 12h and 24h time point. Results shown were mean of 4 biological replicates and tabulated against cellular proteins and expressed in µmoles/ mg protein. The graph from figure 4.2.1 demonstrates that 10µM menadione and acute spiking of 50µM menadione for 15min led to significant increases in the amount of GSH from the 9 h time point to 24h time point. The untreated control at all timepoints shows consistent GSH levels at about 1 µmol/mg protein. The intracellular GSH levels were about 4 fold higher compared to untreated control at 9h and 12h time points. At 24h GSH was approximately 14 times higher compared to control with 10µM menadione addition and 10 times higher when treated with 50µM menadione for 15 min. 51 Figure 4.2.1 showed the amount of intracellular GSH upon menadione treatment over time. K3-50 µM 15 min refers to 50 µM of menadione treated in growth medium with serum for 15 min before changing to serum- free growth medium. K3-10µM refers to treatment with 10µM menadione. Data are shown as mean  SE, where n= 4. * = P [...]... its activity varying with cell type and stages of differentiation99-103 Even though the exact molecular mechanism of GGT action is not thoroughly understood, it is known that GGT transfers the glutamyl moiety to another amino acid, an acceptor The reaction of GGT appears in this general form: Gamma- glutamyl-X + acceptor  Gamma- glutamyl-acceptor + X A wide range of compounds can be used as a gamma- glutamyl... metabolism of GSH81 1.7.2 Protein The translation regulation by the 5’ untranslated region of a GGT mRNA, was initially found using HepG2 cells, and appears to serve as a tissue- specific translational enhancer82 Gamma- glutamyltransferase gene is translated into a single precursor protein that is catalytically inactive83, which undergoes an autocatalytic process into a heavy and light chain84 The heavy chain,... increase the formation of O2 and H2O2 in rat hepatocytes This also resulted in cell membrane blebbing and increased intracellular free Ca2+ due to oxidative stress6 9 Menadione is able to cause DNA damage indirectly via Ca2+- dependent nucleases68 Figure 1.6.2.1 Structure of menadione, taken from Helmenstine A. M66 1.7 Gamma glutamyltransferase 1.7.1 Gene 18 Gamma glutamyltransferase (E.C.3.2.2.2) is an... Glutathione also plays an important role in 11 protecting against ionizing radiation34, assists in supplying copper to CuZnSOD35 and acts as a cofactor for enzymes in different metabolic pathways36, 37 Glutathione also plays a part in protein folding as well as degradation with the cleavage of disulphide bonds, of which insulin is an example38 Glutathione is known to behave as an antioxidant by scavenging... be used as a gamma- glutamyl donor or as acceptor The most natural substrate is GSH (gamma- glutamyl cysteinyl glycine) and the most active and common acceptor substrate is glycylgycine94 Artificial substrates such as gamma- glutamyl-p-nitroanilide and gamma- glutamyl-3-carboxy-4-nitroanilide were developed for the measurements of GGT activity GGT activity is usually measured by a kinetic spectrophotometric... liver cancer High level of GGT was found in non-alcoholic steatohepatitis (NASH), which is a hepatic disease associated with diabetics and obesity, without the alcohol127-129 Clinical studies have also shown a high prevalence of abnormal serum GGT in patients with primary or secondary liver cancer A number of experimental studies have been performed to understand if the GGT isoforms have any relationship... samples showed that there was a greater proportionate increase in GGT than in aspartate transaminase (AST) or alanine aminotransferase (ALT) as shown in figure 1.10.3.1 below 26 Figure 1.10.3.1 The graph was taken from Nagaya et al.139 and the result from serum samples showed the relationship between alcohol intake and average value of GGT, AST and ALT in middle- aged Japanese men The error bars refer to... bronchial and ovarian epithelial tissues showed no staining for GGT108 Hanigan et al have also reported that a decreasing amount of immunoactivity of GGT is observed from normal breast tissues to benign and malignant tissues progressively109 However, another study with ovarian cells showed opposite effect, i.e increasing amount of GGT immunoactivity changing from normal tissues – benign- malignant cells... interval The increase in GGT enzyme activity is much greater than that of AST and ALT 1.10.4 Mechanism of GGT increase in liver disease The mechanism of the increase in serum GGT in liver diseases is poorly understood However Teschke et al.140 found that normal human serum and hepatic GGT is 20fold higher than normal rats Satoh et al have also found that higher values of GGT 27 were seen in patients with... which has the aminoterminal sequence, has a single intracellular transmembrane domain that anchors itself to the cell membrane The light chain, which contains the active site, is situated in the extracellular domain The heavy chain not only holds the light chain to the cell membrane, but also modifies its catalytic enzymatic activity84 There are as many as eight potential sites for glycosylation and the ... form: Gamma- glutamyl-X + acceptor  Gamma- glutamyl-acceptor + X A wide range of compounds can be used as a gamma- glutamyl donor or as acceptor The most natural substrate is GSH (gamma- glutamyl... MTS assay manual) The absorbance of formazan was measured at 490nm This assay measures the dehydrogenase enzyme activity found in metabolically active cells and procedures of this assay were carried... translation regulation by the 5’ untranslated region of a GGT mRNA, was initially found using HepG2 cells, and appears to serve as a tissue- specific translational enhancer82 Gamma- glutamyltransferase