Enzymes Gamma-glutamyl Transpeptidase Research Articles

Changes in Selected Biochemical Markers of Honey Bees Exposed to Fermented Common Tansy Solution (Tanacetum vulgare L.).

Honey bees use pollen and nectar from flowers to produce food. Because they often forage on crops, they are at risk of being exposed to plant protection products (PPPs), both directly and in stored food. Due to the adverse effects of synthetic PPPs on pollinators, biopesticides may be a viable alternative. Common tansy extract is used as one of the natural substitutes for synthetic pesticides. In our study, the effect of fermented common tansy extract on aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transpeptidase (GGTP) activity and the concentration of triglycerides (TGs), total protein (TP), total antioxidant status (TAS), and glucose in honey bee workers' hemolymph was assessed. These biochemical markers give valuable information about the immunity, detoxification, and nutrition of a bee's body. Caged bees were given tansy extract added at various concentrations in sugar syrup for 24 h. Then, they were provided with only sugar syrup. After 7 days of the experiment, hemolymph was collected and analyzed. We observed changes in the activity of AST, ALT, GGTP enzymes and TG, TP, and glucose levels, but not all changes were statistically significant. In terms of AST activity, statistically significant differences were found. All groups tested, including the negative control group, showed reduced enzyme activity values compared to the positive control group. In TG concentration, differences were observed between the groups receiving 2% extract and 1% ethanol. Glucose levels differed between the groups receiving 1% extract and 2% extract and between the positive control group and 1% extract. Bee body proper functioning is affected by changes in enzyme activity, especially those responsible for immunity and detoxification, such as AST, ALT, ALP, and GGTP. Despite the short time of bees' exposure to the agent, the results of study show visible effects. Our results provide a basis for further research on the impact of tansy extract on honey bees.

Mancozeb-induced hepatotoxicity: protective role of curcumin in rat animal model.

Mancozeb-a widely used fungicide in the agricultural sector-is believed to cause toxicity by increasing oxidative stress. This work investigated the efficacy of curcumin in protecting mancozeb-induced hepatotoxicity. Mature Wistar rats were assigned into 4 equal groups: control, mancozeb (30mg/kg/day, ip), curcumin (100mg/kg/day, po), and mancozeb+curcumin. The experiment lasted for 10days. Our results reported that mancozeb elevated aspartate transaminase, alanine transaminase, alkaline phosphatase, lactate dehydrogenase, gamma glutamyltranspeptidase enzyme activities, and total bilirubin level in plasma; and decreased total protein and albumin levels, compared with the control group (P<0.05-0.001). Hepatic tissue levels of malondialdehyde, and advanced oxidation protein products were significantly increased; whereas activities of superoxide dismutase, catalase, glutathione peroxidase, as well as levels of reduced glutathione, vitamin C, and total protein were reduced (P<0.05-0.001). Histopathological examination showed marked histological changes. Co-treatment with curcumin improved the antioxidant activity; reversed oxidative stress and biochemical changes; and restored most of the liver histo-morphological alterations; thus, attenuating the hepatic toxicities induced by mancozeb. These results indicated that curcumin could protect against detrimental hepatic effects induced by mancozeb.

Synthesis, structural and spectral characteristics of novel n,π-chelate complexes of Pd(II) and Pt(II) with N-allylthioureas and their influence on the growth of spheroids cells MCF-7 and GGT activity

Four mononuclear n,π-chelate complexes [M(HL1,2)Cl2] (M = Pd2+, Pt2+) have been obtained by the reactions of PdCl2 or K2[PtCl4] with N,N-diethyl-N'-(2-propenyl)thiourea (HL1) or N-cyclohexyl-N'-(2-propenyl)thiourea (HL2) in the presence of HCl. According to X-ray diffraction study, the molecules of HL1,2 are coordinated in a bidentate manner with the formation of six-membered chelate metallocycles in a twist-boat conformation. The diagonal arrangement of “soft-hard” donor atoms (S–M−Cl, C–M−Cl) in the coordination sphere leads to the Pearson's effect of “molecular antisymbiosis in the trans effect” that causes the formation of complexes with an M:L ratio of 1:1. However, interaction in the ratio 1:2, 1:3 is also possible in the solution under the conditions of monodentate coordination of HL1 and HL2. All compounds were investigated by IR, UV–vis and NMR spectra. The 1H/13C NMR study showed that this method is reliable for establishing the formation of a π-coordinating bond. The similarity of the synthesized complexes structure with cisplatin implies the same mechanism of their antitumor action. Thus, it was investigated their effect on the growth and biochemical characteristics of human breast adenocarcinoma cells MCF-7 (grown in three-dimensional culture as spheroids) and the activity of the enzyme gamma-glutamyl transpeptidase (GGT). The results showed that all studied π-complexes are more active compared to cisplatin, and they exhibit pronounced anti-metastatic and pro-apoptotic activity, which one is caused by their inhibitory effect.

Mechanistic role of astaxanthin derived from shrimp against certain metabolic disorders.

Oxidative stress caused by the imbalance between production of oxidants and antioxidants in the body leads to the development of different ailments. The bioactive compounds derived from marine sources are considered to be safe and appropriate to use. Astaxanthin possesses antioxidant activity about 100–500 times higher than other antioxidants such as α‐tocopherol and β‐carotene. It has numerous health benefits and vital pharmacological properties for the treatment of diseases like diabetes, hypertension, cancer, heart disease, ischemia, neurological disorders, and potential role in liver enzyme gamma‐glutamyl transpeptidase which has significance in medicine as a diagnostic marker. The primary source of astaxanthin among crustaceans is shrimps and the presence of astaxanthin protects shrimps from oxidation of polyunsaturated fatty acids and cholesterol. Conclusively, astaxanthin derived from shrimps is very effective against oxidative stress which can lead to certain ailments.

Usefulness of gamma-glutamyl transpeptidase and mean corpuscular volume in alcoholic liver disease

Introduction: Excessive consumption of alcohol and alcoholism are widely observed risk factors for damage to health and social problems. Early identification of alcoholic liver disease could improve the possibility of early treatment and reduce damage to health and health costs. Our present study was anticipated to establish the association between the liver enzyme gamma-glutamyl transpeptidase and an haematocrit parameter mean corpuscular volume in alcohol liver disease patients. Materials and Methods: Our study includes 100 subjects, with 50 cases and 50 controls. Serum was collected for the analyses of GGT, liver enzymes ALP, AST, ALT, total protein, albumin, bilirubin. Haemoglobin, Mean corpuscular volume, PCV and RBC were also analyzed. The statistical relationship between AST and ALT has been calculated. Statistical data analysis was performed by SPSS 20 software. Results: The values of the GGT and MCV levels were significantly increased in patients compared to controls (p Conclusion: The Overall verdict of this study shows that the increase in ?GT and MCV levels in ALD patients can be used for added diagnostic markers for the diagnosis of alcoholic liver disease. Keywords: Gamma glutamyl transpeptidase (GGT), Mean corpuscular volume (MCV), Alcoholic liver disease (ALD).

Amlodipine alleviates cisplatin-induced nephrotoxicity in rats through gamma-glutamyl transpeptidase (GGT) enzyme inhibition, associated with regulation of Nrf2/HO-1, MAPK/NF-κB, and Bax/Bcl-2 signaling

BackgroundThe therapeutic utility of the effective chemotherapeutic agent cisplatin is hampered by its nephrotoxic effect. We aimed from the current study to examine the possible protective effects of amlodipine through gamma-glutamyl transpeptidase (GGT) enzyme inhibition against cisplatin nephrotoxicity. MethodsAmlodipine (5 mg/kg, po) was administered to rats for 14 successive days. On the 10th day, nephrotoxicity was induced by a single dose of cisplatin (6.5 mg/kg, ip). On the last day, blood samples were collected for estimation of kidney function, while kidney samples were used for determination of GGT activity, oxidative stress, inflammatory, and apoptotic markers, along with histopathological evaluation. ResultsAmlodipine alleviated renal injury that was manifested by significantly diminished serum creatinine and blood urea nitrogen levels, compared to cisplatin group. Amlodipine inhibited GGT enzyme, which participates in the metabolism of extracellular glutathione (GSH) and platinum-GSH-conjugates to a reactive toxic thiol. Besides, amlodipine diminished mRNA expression of NADPH oxidase in the kidney, while enhanced the anti-oxidant defense by activating Nrf2/HO-1 signaling. Additionally, it showed marked anti-inflammatory response by reducing expressions of p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-κB), with subsequent down-regulation of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1). Moreover, amlodipine reduced Bax/Bcl-2 ratio and elevated hepatocyte growth factor (HGF), thus favoring renal cell survival. ConclusionsEffective GGT inhibition by amlodipine associated with enhancement of anti-oxidant defense and suppression of inflammatory signaling and apoptosis support our suggestion that amlodipine could replace toxic GGT inhibitors in protection against cisplatin nephrotoxicity.

PHARMACOLOGICAL EVALUATION OF BETA‐HYDROXIPHOSPHOCARNITINE IN NO‐ALCOHOLIC ESTEATOHEPATITIS INDUCED IN RATS

ObjectiveTo evaluate the pharmacological effect of Β‐Hydroxyphosphocarnitine (β‐HFC) on non‐alcoholic steatohepatitis induced in rats.IntroductionNon‐alcoholic fatty liver disease (EHGNA) is a disease with high prevalence, usually accompanied by metabolic syndrome. Currently the treatment to EHGNA is pioglitazone. However, it is reported that pioglitazone causes several side effects reason to stop treatment. L‐carnitine has pharmacological effect in the metabolic syndrome, but it is poorly absorbed. The β‐HFC is an analogue of L‐carnitine that has shown effect on metabolic síndrome and maintaining its pharmacological properties.MethodEHGNA was developed in male Wistar rats with a mixed diet based on coconut oil, carbon tetrachloride and high fructose drink. The treatments were as follows: Without treatment, pioglitazone and β‐HFC in healthy groups and with EHGNA. Histological sections of the liver were made and stained with Masson's Trichromic Stain to see collagen fibers and Hematoxylin &amp; Eosin to see hepatocyte morphology. Quantities of liver enzymes Aspartate aminotransferase (AST), alanine aminotransferase (ALT), Gamma glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) and triglycerides were performed.ResultsIn the histology of the healthy control rats, the hepatocyte architecture is well observed, no liver damage characteristics are observed, for example: fat drops, inflammatory cells, necrosis, apoptosis and hepatocytes in the balloon. Healthy groups administered with pioglitazone and β‐HFC as well as the control show no characteristics of liver damage. Histologies of rats with EHGNA showed the characteristics of liver damage and greater amount of collagen fibers. Rats with steatophepatitis treated with β‐HFC showed reduced hepatocyte damage and fibrosis compared to EHGNA control rats. Liver enzyme levels increased 2.5 times more in the steatohepatitis group compared to those in control rats. Liver enzyme levels showed considerable reduction in those rats with statohepatitis and were treated with β‐HFC, showing values for the enzymes ALT, AST, GGT and ALP 85.79, 101.3, 3.2 and 556.4 U/L, respectively for group with steatohepatitis and 74.34, 77.7, 1.0 and 327 U/L, respectively for the group with steatohepatitis treated with β‐HFC. Triglyceride levels increased in rats with steatohepatitis compared to those in control rats and reduction in levels was observed for those who were treated with β‐HFC showing values of 63.7 (control), 255.1 (steatohepatitis) and 48.4 mg/dL (steatohepatitis + β‐HFC). The same behavior showed liver levels 0.04, 0.14 and 0.04 mg/dL respectively.ConclusionB‐HFC reduced liver damage levels in rats with EHGNA.

A Correlative Study Between Haematological and Biochemical Parameters in Hepatitis B

Hepatitis-B (HBV) is a viral disease cause liver damage, cirrhosis, fibrosis and hepatocellular carcinoma. Present study attempted to elucidate the biochemical and haematological markers other than Australia antigen, of hepatitis,B,vairusV (HBsAg) for better assessment of HBV infection. The present study was conducted on 76 men, 50 of them were found to be HBeAg positive and 26 were negative, mean age was53±5.7years. Haematological parameters such as Absolute Erythrocyte( Abs Eryt), Absolute Leukocyte(Abs Leuk) , Haemoglobin(Hb), Packed Cell Volume(PCV),Mean Corpuscular Volume (MCV), Red Cell Distribution Width (RDW), Mean Corpuscular Haemoglobin (MCH),MCH Concentration(MCHC) ,Neutrophils(Neut) ,Lymphocyte(Lymph), Monocyte(Mono), Eosinophil(Eosin) , Basophil (Baso) , Absolut platelet(Abs.Plt), Red Blood Distribution(RBD)and biochemical markers such as Alanine aminotransferase(ALT), Aspartate aminotransferase(AST), Alkaline phosphatase(ALP), Gamma glutamyl transpeptidase(GGT), Total Bilirubin (T.Bil), Albumin(Alb), C- reactive protein (CRP), Amylase(Amy), Creatinine(Crea), Sodium(Na) , Potassium(K), were estimated for HBV patients and healthy groups.Statically at (P≤ 0.05) Abs Plt was highly significant elevated, Hb, Abs Leuk, Neut%, Lymph% and Eosin% were significant increases while other haematological parameters showed no differences in HBV patients compared with controls. Liver enzymes (ALT, AST, GGT) and T.Bil were highly significant increased, Alb , CRP and Amyl were moderately increased, niether Crea, Na nor K levels have differences in HBV patients compared with controls.ALT has strong positive correlation with Leuk and with Abs Plt in HBV patients. liver enzymes ALT, AST, ALP, GGT, T.Bil and Abs Plt can be used as monitoring markers with the strong correlation between ALT and Abs Plt as an assessment tools for HBV infection.

Human Pluripotent Stem Cell-Derived Kidney Model for Nephrotoxicity Studies.

Current in vitro models for identifying nephrotoxins are poorly predictive. We differentiated human pluripotent stem cells (hPSCs) into three-dimensional, multicellular structures containing proximal tubule cells (PTCs) and podocytes and evaluated them as a platform for predicting nephrotoxicity. The PTCs showed megalin-dependent, cubilin-mediated endocytosis of fluorescently labeled dextran and active gamma-glutamyl transpeptidase enzymes. Transporters from both the ATP-binding cassette (ABC) and the solute carrier (SLC) families were present at physiological levels in the differentiated cells, but important renal transporters such as organic anion transporter 1 (OAT1), OAT3, and organic cation transporter 2 (OCT2) were present only at lower levels. Radioactive uptake studies confirmed the functional activity of organic cation transporter, novel, type 2 (OCTN2), organic anion transporter polypeptide 4C1 (OATP4C1), and OCTs/multidrug and toxin extrusion proteins (MATEs). When treated with 10 pharmacologic agents as a test of the platform, the known nephrotoxic compounds were distinguished from the more benign compounds by an increase in tubular (PTC, kidney injury molecule 1 (KIM-1), and heme oxygenase 1 (HO-1)) and glomerular (nephrin [NPHS1]/Wilms tumor protein [WT1]) markers associated with nephrotoxicity, and we were able to distinguish the type of nephrotoxin by examining the relative levels of these markers. Given the functions demonstrated and with improved expression of key renal transporters, this hPSC-derived in vitro kidney model shows promise as a platform for detection of mechanistically different nephrotoxins.

Structure of 6-diazo-5-oxo-norleucine-bound human gamma-glutamyl transpeptidase 1, a novel mechanism of inactivation.

Intense efforts are underway to identify inhibitors of the enzyme gamma-glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma-glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6-diazo-5-oxo-norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM-1 min-1 and the Ki was 2.7 ± 0.7 mM. The crystal structure of DON-inactivated hGGT1 contained a molecule of DON without the diazo-nitrogen atoms in the active site. The overall structure of the hGGT1-DON complex resembled the structure of the apo-enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1-DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α-amine of Thr381. The structure of DON-bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.

Nephroprotective Hepatoprotective Potential and Antioxidant Role of Carob Pods (Cerotonia siliqua L.) against Carbon Tetrachloride-induced Toxicity in Rats

Abstract: Objective: The aims of current study are the evaluation of the protective effect and antioxidant role of carob pods against carbon tetrachloride (CCl4)-induced oxidative stress, hepatotoxicity and nephropathy. Material and Methods: The present experiment was designed as I (control), II (0.5 ml/kg CCl4), III (%10 CP), IV (CCl4 0.5 ml/kg+%10 CP) groups. While rats in group I and III were fed with a diet without CCl4, II, and IV groups received twice 0.5 ml/kg/week, where IV group additionally received %10 CP supplementation for 50 days. The protective roles and antioxidant activity of the CP supplementation feed against CCl4-induced oxidative stress and toxicity were evaluated by histopathological changes, measuring hepatic and renal damage biomarkers (HRDB), antioxidant defence system constituents (ADSC) and malondialdehyde (MDA) parameters in the erythrocyte, liver, brain, kidney and spleen tissues of rats. Results: According to the results, the biochemical analysis showed a considerable increase in the serum AST (aspartate aminotransferase), ALT (alanin aminotransferase), GGT (gamma glutamyl transpeptidase) and LDH (lactate dehydrogenase) enzymes, creatinine and urea, and decrease in the group II as compared to that of I group. On contrary, such parameters were decreased in IV group as compared to that of group II. In addition, the results showed that CP supplementation diet restored the CCl4 induced MDA (malondialdehyde) and ADSC towards to control. The hepatoprotection of CP is further substantiated by the almost normal histological findings in IV group against degenerative changes in II group. Protective effects by CP are further substantiated by the almost normal HRDB for kidney and liver in IV treated group as against degenerative changes in the II treated rats. Conclusion: The results indicated that CP could be as an important as diet-derived antioxidants in preventing oxidative damage in the tissues by reducing the MDA or inhibiting the production of CCl4-induced free radicals and liver and kidney destruction. Key words: Carbon tetrachloride, Carob pods, Protective potential, Antioxidant role.

Thermo- and salt-tolerant chitosan cross-linked γ-glutamyl transpeptidase from Bacillus licheniformis ER15

Gamma-glutamyl transpeptidase enzyme, from Bacillus licheniformis ER15 (BLGGT), was produced extracellularly using a complex medium with high enzyme titers. Enzyme was concentrated and purified using ultra-filtration and ion exchange chromatography, respectively, with a purification fold of 4.6 and 50.11% yield. Enzyme was covalently immobilized onto chitosan microspheres (CMS). Immobilization was standardized with respect to pH, enzyme load and time. Immobilization efficiency of 11.9U/mg dry weight of microsphere was obtained in Tris-HCl buffer (pH 9.0) at 18°C in 4h. Immobilized enzyme (CMS-GGT) exhibited improved thermal stability (t1/2 of 70.7min at 60°C), activity in a broader pH range and improved salt stability in 18% (3M) sodium chloride solution as compared to free enzyme. Both free and immobilized enzymes specifically converted glutamine to glutamic acid in a mixture of amino acids. CMS-GGT had a better shelf life and high recyclability retaining 90% catalytic efficiency upto 10 reaction cycles. For long-term storage, CMS-GGT can be disinfected using either sodium azide or sodium hypochlorite solution without affecting enzyme activity. Thus, the present study provides an easy and efficient method for GGT enzyme immobilization that results in an improved and robust enzyme preparation.

Abstract 4546: Depleting cysteine: A novel approach to tumor therapy

Abstract A cure remains elusive for many types of tumors that initially respond to chemotherapy but recur with drug-resistant disease. Cysteine, (or its oxidized form cystine) is an essential amino acid for tumors. Enhanced uptake of cysteine and/or cystine is required for tumors to maintain their intracellular glutathione (GSH) levels and redox balance. In addition, some stem cells within epithelial tumors express CD44v8-10, which stabilizes the cystine transporter, increasing cystine uptake into the cell and enhancing resistance to pro-oxidant therapy. Depleting cysteine during treatment has a profound effect on sensitizing the tumor to therapy. We have a novel strategy that reduces the cysteine (and cystine) concentration in serum and extracellular fluid. The strategy exploits the finding that a severe cysteine deficiency develops in mice lacking the enzyme gamma-glutamyl transpeptidase (GGT). This enzyme reclaims the cysteine from GSH as the blood is filtered through the kidney. Inhibiting GGT activity results in excretion of GSH in the urine and a precipitous drop in the concentration of cysteine and cystine in serum. The inhibitors of GGT that have been evaluated in the clinic are glutamate analogs and are too toxic for clinical use. We have discovered and are developing a new class of GGT inhibitors that are not glutamate analogs. These inhibitors are orders of magnitude less toxic than the glutamate analogs. We have determined the crystal structure of human GGT. In the current study we have co-crystallized human GGT with a series of inhibitors. We report the identification of residues within the active site that are critical for inhibitor binding. We have identified changes in the conformation of the active site upon substrate binding. We have also delineated movement within the active site during substrate cleavage. Site-directed mutagenesis and functional assays have confirmed the role of the key residues identified in the structural studies. These data provide new insights for continued development of a potent, specific, non-toxic inhibitor of GGT that can be used clinically in cancer therapy. Supported by an Institutional Development Award (IDeA) from the National Institutes of Health under grant P20GM103640. Citation Format: Marie H. Hanigan, Anthony W. G. Burgett, Nancy Wakeham, Simon S. Terzyan. Depleting cysteine: A novel approach to tumor therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4546. doi:10.1158/1538-7445.AM2015-4546

Biochemical characterization of preovulatory and cystic ovarian follicular fluid of sow

Aim: The present study was designed to biochemically characterize ovarian cyst follicular fluid of sows and to relate possible changes in relation to preovulatory ovarian follicular fluid of sows. Materials and Methods: Ovaries were collected from adult and non-pregnant slaughtered sow (Hampshire X local cross). A total of 16 pairs of ovaries were investigated, and they were classified according to their pathological and physiological status into two categories i.e. preovulatory and cystic follicle (diameter >25 mm with turbid appearance). Fluid was aspirated from the follicles and stored at −20°C prior to assaying. Follicular fluid samples were analyzed for glucose, total protein, cholesterol, triglycerides, blood urea nitrogen (BUN), urea, creatinine, uric acid. The activities of the enzymes aspartate transaminase, alanine transaminase (ALT), alkaline phosphatase (ALP), creatine kinase (CK) and gamma-glutamyl transpeptidase (GGT) were also estimated by spectrophotometric methods. The data were statistically analyzed using the SPSS software. Results: As follicles became cystic, the concentrations of glucose, cholesterol, triglyceride, total protein and albumin showed a significant (p<0.05) increased while BUN decreased significantly (p<0.05). The enzyme activities of ALT, ALP and CK increased significantly (p<0.05), but the activity of GGT significantly (p<0.05) decreased. Conclusion: Results obtained showed that there was a significant change in most of the cystic follicular fluid metabolites.

Abstract 683: Sensitizing tumors to pro-oxidant therapy by inhibiting gamma-glutamyl transpeptidase

Abstract We have reported that 73% of ovarian tumors express the cell surface enzyme gamma-glutamyl transpeptidase (GGT). Clinical studies have shown a poorer prognosis for ovarian tumors that are GGT-positive compared to GGT-negative tumors. GGT-positive tumors are able to use the glutathione in the interstitial fluid as an additional source of cysteine, the limiting component of intracellular glutathione synthesis in tumors. The concentration of cysteine/cystine in standard tissue culture media is more than 3 times the concentration in interstitial fluid. In this study we show that in studies of cultured cells, the high concentration of cysteine in tissue culture media obscured the role of GGT expression in drug resistance. However, culturing human ovarian tumor cells in tissue culture media containing physiological concentrations of cystine and glutathione demonstrated that the supply of cysteine/cystine regulates intracellular glutathione concentrations. In these studies, inhibition of GGT blocked the use of extracellular glutathione as a source of cysteine and sensitized the tumors to cisplatin, carboplatin and melphalan. Inhibiting GGT in patients would also inhibit GGT in the kidney resulting in the excretion of glutathione in the urine and depletion of cysteine from serum and interstitial fluid. As a result, inhibition of GGT in patients would sensitize both GGT-positive and GGT-negative tumors to pro-oxidant chemotherapy. Clinical application of this therapeutic strategy is limited by the fact that most inhibitors of GGT are glutamate analogs and too toxic for use in humans. We discovered a non-glutamate analog that is an uncompetitive inhibitor of GGT, and are developing more potent, non-toxic GGT inhibitors. We recently published the crystal structure of human GGT. We have now also solved the structure of the substrate-bound enzyme, the form of the enzyme to which our uncompetitive inhibitors bind. Using structure-based drug design and our physiologically relevant tissue culture system we are designing new GGT inhibitors and evaluating them for their ability to sensitize tumors to chemotherapy regimens used in the clinic. Supported by an Institutional Development Award (IDeA) from the National Institutes of Health under grant P20GM103640. Citation Format: Marie H. Hanigan, Nancy Wakeham, Stephanie Wickham, Simon S. Terzyan. Sensitizing tumors to pro-oxidant therapy by inhibiting gamma-glutamyl transpeptidase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 683. doi:10.1158/1538-7445.AM2014-683

False positivity of gamma-glutamyl transpeptidase measurement in urine

Although enzymuria tends to be associated to renal injury, there are no studies that have evaluated the presence of the enzyme gamma-glutamyl transpeptidase (GGT) spectrophotometry in the urine using a non-nephrotoxic agent (Nerium oleander) in order to evaluate the possibility of false positive results. The urinary GGT/urinary creatinine concentration ratio (uGGT/uCr) of 10 healthy dogs was calculated and posteriorly confronted with data from clinical evaluation, hematological and serum biochemical profiles, creatinine clearance (CrC), urinalysis, urine protein/creatinine ratio (UPC), electrocardiogram, systemic blood pressure (SBP) and light and electron microscopy. The results for kidney histology, SBP, UPC and CrC were not significantly different in any of the time-points analyzed. However, uGGT/uCr was significantly higher when measured 4 hours and 24 hours after administration of N. oleander. The measurement of the urinary GGT enzyme, as performed in many studies, yielded false positive results in dogs poisoned by a non-nephrotoxic agent.

Synthetic Pyrethroid effect on blood plasma biomarker enzymes and histological changes in Catla catla

Alpha-cypermethrin is an isoform of cypermethrin; it is an active pyrethroid used extensively to control a wide range of pests in agriculture and animal breeding. In this study four groups of six fish were examined. The first group served as a control in fresh water alone, with no pyrethroid. The second, third and fourth groups were exposed to alpha-cypermethrin for 4, 8 and 96 h respectively. At the end of the each exposure period, the fish were sacrificed, and the required muscle tissues were collected for histological examination. The blood was drawn with heparinized needles and processed for serum enzymatic studies. Serum enzymes such as aspartate transaminase (AST), alanine transaminase (ALT), amylase, acid phosphatase (ACP) and gamma-glutamyl transpeptidase (GGT) were measured at 4, 8 and 96 h. AST enzyme activity was significantly increased at 4 h, whereas ALT and amylase enzyme activities were significantly reduced at all the time points. ACP enzyme activity was significantly reduced at 4 and 8 h, whereas GGT enzyme activity was significantly increased at all the time points. Hepatocyte cytoplasmic vacuolisation and degeneration, rupture of blood vessels, and necrosis was found at all time points. Congestion of blood vessels, bulging, distortion of filaments, erosion and disintegration of blood corpuscles and hyperplasia of epithelium were found in treated gills at 4, 8 and 96 h. Breakdown of muscle fibres, vacuolation and accumulation of lipids and melanin in white muscle were observed in treated fish muscle at 4, 8 and 96 h.

Uticaj profesionalne izloženosti pesticidima i konzumiranja alkoholnih pića na funkcije jetre

Various pesticides are applied in agriculture for crop protection. They could have toxic effects on nervous system, liver, kidneys and the other organs. The aim of the study was to investigate effects of occupational exposure to pesticides and consumption of alcoholic beverages on liver functions in agricultural workers. The study included sample of 117 male agricultural workers involved as tractor drivers in pesticide application at the workplace and the control group of 60 tractor drivers who do not applied pesticides. We collected data concerning age, use of alcohol and duration of exposure to pesticides. Parameters of liver functions in serum were determined in both groups of workers. In agricultural workers exposed to pesticides, activities of enzymes gamma-glutamyl transpeptidase, alanine and aspartate aminotransferases were higher comparing to the control group, but these differences were not statistically significant. Consumption of alcohol statistically significantly increased activity of gamma-glutamyl transpeptidase in both groups of workers. The most important measures concerning health protection of agricultural workers exposed to pesticides are continuous implementation of the safety measures at work, health promotion at workplace and regular health surveillance in occupational health services for the purpose of early detection of worker's health disorders.

Abstract P005: Traditional Cardiovascular Risk Factors Explain the Association Between GGT and Incident Cardiovascular Events, but not Total Mortality

Background: Elevated levels of the liver enzyme gamma glutamyl transpeptidase (GGT) are associated with incident diabetes and the metabolic syndrome, but there is conflicting evidence regarding the association between GGT and cardiovascular risk. Hypothesis: GGT is positively associated with risk of coronary heart disease (CHD), congestive heart failure (CHF), and total mortality, independently of metabolic factors. Methods: We analyzed data from 9,104 participants in the Atherosclerosis Risk in Communities (ARIC) Study without cardiovascular disease or high alcohol consumption (&gt;2 drinks per day for women and &gt; 1 drink per day for men) at baseline (Visit 4, 1996-1999). GGT was measured from thawed plasma samples from Visit 4. The primary outcome was incident CHD (non-fatal MI, coronary revascularizations, or CHD death). Secondary outcomes were CHF and total mortality. We estimated the hazard ratios from Cox models for the risks associated with quartiles of GGT and ln-transformed GGT. Results: During a median of 10 years of follow-up, there were 925 CHD events, 654 CHF events, and 1,084 deaths. Male gender, diabetes, and components of the metabolic syndrome were associated with higher GGT levels at baseline. GGT was positively associated with risk of CHD and CHF in age-adjusted models, but not after adjustment for components of the metabolic syndrome and diabetes ( Table ). In contrast, the highest quartile of GGT remained associated with total mortality even after adjustment for all metabolic factors. Similar findings were seen for analyses of ln-GTT. Conclusion: The association between GGT and incident cardiovascular events was largely explained by metabolic risk factors. However, elevated GGT was associated with total mortality independent of the metabolic syndrome and diabetes, suggesting a potential alternative mechanism of risk. Table Hazard Ratios of Incident CHD, CHF and Total Mortality Associated with Quartiles * of GGT GGT Q1 (≤14 U/L)n=2,039) GGT Q2 (15-20 U/L)n=2,301 GGT Q3 (21-30 U/L)n=2,385 GGT Q4 (≥31 U/L)n=2,379 CHD Model 1 Ref 1.16(0.93-1.44) 1.28(1.04-1.58) 1.62(1.32-1.99) Model 2 Ref 1.10(0.89-1.37) 1.18(0.95-1.47) 1.47(1.19-1.82) Model 3 Ref 1.03(0.82-1.28) 1.06(0.85-1.32) 1.30(1.04-.1.61) Model 4 Ref 1.00(0.80-1.24) 1.00(0.80-1.24) 1.17(0.94-1.46) CHF Model 1 Ref 1.12(0.86-1.45) 1.36(1.06-1.74) 1.97(1.55-2.51) Model 2 Ref 1.00(0.77-1.29) 1.12(0.87-1.45) 1.59(1.24-2.02) Model 3 Ref 0.96(0.74-1.24) 1.04(0.80-1.35) 1.41(1.09-1.81) Model 4 Ref 0.90(0.70-1.18) 0.93(0.71-1.20) 1.17(0.91-1.51) TOTAL MORTALITY Ref 0.90(0.69-1.17) 0.94(0.72-1.22) 1.13(0.87-1.46) Model 1 Ref 1.14(0.94-1.38) 1.16(0.96-1.41) 1.52(1.26-1.83) Model 2 Ref 1.14(0.94-1.39) 1.15(0.94-1.40) 1.50(1.24-1.83) Model 3 Ref 1.16(0.95-1.41) 1.16(0.95-1.42) 1.48(1.21-1.80) Model 4 Ref 1.13(0.93-1.38) 1.10(0.90-1.35) 1.34(1.09-1.64) * Quartiles defined on the entire study population Model 1: Adjusted for age, race, and sex Model 2: Adjusted for Model 1 + BMI and smoking Model 3: Adjusted for Model 2 + metabolic syndrome components (fasting hyperglycemia (BS&gt;100), hypertension (SBP&gt;130, DBP&gt;85 or anti-hypertension medications), hypertriglyceridemia (&gt;150 mg/dl), low HDL (&lt;40 mg/dl for men and &lt;50 mg/dl for women) and abdominal obesity (waist circumference &gt; 102 cm for men and &gt;88 cm for women))Model 4: Adjusted for Model 3 + diabetes mellitus

Cytotoxic Activity of &amp;lt;i&amp;gt;Thelesperma megapotamicum&amp;lt;/i&amp;gt; Organic Fractions against MCF-7 Human Breast Cancer Cell Line

Thelesperma megapotamicum (Asteraceae) is commonly used in Argentine to treat various diseases (renal, digestive affections, and as anaesthesia). The present study showed the mechanisms involved “in vitro” cytotoxicity of T. megapotamicum Fractions. Five Fractions (F1 - F5) were separated by column chromatography (Silica gel) using hexane:diethyl ether as eluents. Viability was evaluated in Human breast carcinoma cell line (MCF-7) by staining with crystal violet. With respect to F1 Fraction treatment, the cell survival was 49.14% ± 8.87%, while the F2 and F3 ones exhibited a strong reduction of cell viability to only 26.35% ± 1.63% and 23.3%1 ± 0.53% of the control cell at 50 μg/ml, respectively. Apoptotic effect of these Fractions was detected using FITC-labeled Annexin V and propidium iodide binding assays and was confirmed by a higher proportion of apoptotic cells due to F2 and F3 treatments. T. megapotamicum active Fractions could facilitate the tumoral cells death by decreasing the activity of the enzyme Gamma-glutamyltranspeptidase and causing alteration in cell membrane sialoglycoconjugates and others involved anticancer mechanisms including apoptosis.