Study of Triclabendazole (TCBZ) Effect on Aspartate Transaminase (AST) Activity of Fasciola gigantica Parasite and Liver Enzyme Activity Assay

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1 2014 Study of Triclabendazole (TCBZ) Effect on Aspartate Transaminase (AST) Activity of Fasciola gigantica Parasite and Liver Enzyme Activity Assay Shima Shafaei 1, Ali Farahnak 1*, Taghi Golmohammadi 2, Mohammad Reza Esharghian 3, Mohammad Bagher Molaei Rad 1 1 Department of Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 2 Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 3 Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. ARTICLE INFO Article type: Original article Keywords: Aspartate Aminotransferase Fasciola Triclabendazole ABSTRACT Background: Aspartate transaminase (AST) is an important enzyme in parasite and liver tissue. The purpose of this investigation is to evaluate triclabendazole (TCBZ) effect on AST activity of Fasciola gigantica parasite. To compare of enzyme level of parasite and its host tissue, enzyme activity of F. gigantica parasite and liver tissues were also determined. Method: The livers were collected from sheep slaughtered in local slaughterhouse and living F. gigantica parasites were isolated. The washed parasites were cultured in buffe rmedia with or without Triclabendazole (Egaten ); 15μg/mL in an incubator at 37 C. Extractions of collected parasites and liver tissues were prepared by homogenizing buffer in a Mortar and pestle. Extraction samples were examined for protein measurement, AST activity assay and protein recognition. Results: The results of AST assay revealed, enzyme activity for treated and untreated is not significant. Healthy liver tissue shows significantly higher enzyme activity than parasite. Enzyme activity for healthy and infected liver tissues was significant. Enzymatic proteins including Cathepsin L & B (Protease) were recognized in parasite samples. Conclusion: Although AST could not be concerned as an indicator for efficiency treatment, however may be involved as a biomarker for biochemical comparison of parasite and host tissue. J Pharm Care 2014; 2 (4): Please cite this paper as: Shafaei S, Farahnak A, Golmohammadi T, Esharghian M, Molaei Rad M. Study of Triclabendazole (TCBZ) Effect on Aspartate Transaminase (AST) Activity of Fasciola Gigantica Parasite and Liver Enzyme Activity Assay. J Pharm Care 2014; 2 (4): Introduction The total number of people that infected with fascioliaisis is estimated 2.4 million in 61 countries and that the number at risk is more than 180 million throughout the world (1). Egaten (Triclabendazole Drug) as the drug of choice for fascioliasis is given orally at a dose of 10 mg/kg (2). The studies have been * Corresponding Author: Address: Dr Ali Farahnak Department of Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Enghelab Square, Tehran, Iran. Tel: , Fax: farahnak@tums.ac.ir suggested that tubulin organization was disturbed in the tegument of triclabendazole (TCBZ)-susceptible flukes (3). Helminthes are capable to catabolism amino acids by comparable pathways to those discovered in mammals. There are, however, dissimilarity in detail between the pathways of amino acid metabolism in helminthes and mammals, predominantly in the metabolism of the sulphur amino acids, arginine and proline (4). AST is dimeric enzyme with molecular weight of almost 45 kda. This enzyme catalyzes the reaction of aspartate and α-ketoglutarate to oxaloacetate and glutamate. AST is commonly considered clinically as a part of diagnostic

2 liver function tests (5). The enzyme has been described from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum (6). However there is no report on enzyme level of F. hepatica and TCBZ effect on it in Iran. In the present work, TCBZ effect on AST enzyme activity of Fasciola gigantica parasite (Iranian isolates) and AST s level of parasite and its host liver tissues are discussed. Materials and Methods Extractions preparation of parasite and liver samples 10 healthy and infected sheep liver tissues with Fasciola gigantica parasites were collected from local abattoir (Soleymani, Tehran, Iran). Fasciola gigantica isolated, identified based on morphological characters and were washed 3-4 times with washing buffer. Collected parasites were cultured in PBS, 7.4 media with 15 μg TCBZ (Egaten ; Swiss, Novartis Pharma AG) per ml solutions or without drug for 4-6 hours in an incubator at 37 C. Recollected parasites and liver tissue were homogenized with 3 volumes of phosphate buffer 7.2, in a Mortar and pestle, The suspension was centrifuged (10000g for 30 min at 4 C) and supernatant was stored at -20 C (8). Enzyme activity assay of sample extractions The Aspartate Transaminase (AST) Enzymatic Assay kit uses a coupled enzymatic reaction scheme: aspartate and a-ketoglutarate are first converted to glutamate and oxaloacetate which is converted by malate dehydrogenase to make malate and NAD+. The conversion of the NADH to NAD+ product, measured at 340 nm, is proportional to the level of AST enzyme in the sample. Enzyme activities of samples were measured according to kit protocol. 1000μL mixed solution (reagent) and 100μL extraction sample of parasite or liver tissue were added into a cuvette and the absorbance of each samples was measured using a spectrophotometer. The concentration of AST in each sample was then directly determined from the change in absorbance at 340 nm within 5 minutes time at 37 C (9). Measurement and recognition of proteins Supernatants of samples were investigated for protein concentration measurement by Bradford method with BSA standard solutions as duplicate. The absorbance of samples were measured at 595 nm(8).sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and coomassie blue staining were used to separate and stain the protein components of samples. Samples were mixed with sample buffer and were run on 10% acrylamide gels. Finally, the gel was stained with coomassie blue R-250. Molecular weights of sample proteins were compared with respect to the protein marker (8). The proteins were recognized by using ratio factor (Rf) of molecular weights and Expasy protein database (10). Detection of significant differences To detect significant differences between treated and untreated parasite enzyme activities and liver tissue, independent two sample t-test was used (11). Results Protein concentration was detected 9.7 mg/ ml and9.2 mg/ml for treated and untreated Fasciola gigantic parasites respectively. AST activity of treated parasite and untreated group is not significant (p>0.05). Liver tissue shows significantly higher enzyme activity than parasite (p<0.05). However, Enzyme activity for healthy and infected liver tissues was significant (p<0.05). The results of the protein concentration and enzyme activities of samples are presented in table 1. Extraction samples were analyzed by SDS-PAGE electrophoresis and the results are shown in figure 1 and 2. SDS-Page gel shows a protein band with 47 kda for AST enzyme in samples. Cathepsin L and Cathepsin B proteins (Enzymes) were identified in parasite samples. The recognized proteins are presented in table 2 and 3. Discussion The obtained bands of protein SDS-PAGE show no significant difference between control and treated sample parasites. Listed proteins acquired from SDS-PAGE of parasite and liver tissue shows their variances (Tables 2 and 3). It seems that, these different proteins including Cathepsin L and Cathepsin B could be used for purified specific antigen of parasite as a source for vaccine synthesis against fascioliasis and diagnosis by the future surveys (12). SDS-Page gel shows a protein band with 47 kda for AST enzyme in parasite and liver samples. More researches are needed to clear of different aspects of these isoenzymes. Our results demonstrate parasite AST activity is significantly higher than liver tissues. According to published results, the mammalian tissues and mature Fasciola were fractionated into nucleic, mitochondrial and cytosolic by differential centrifugation and the activity and distribution of AST(EC, ) of the different fractions were measured. Isozymes pattern of them were also determined by DEAE-Cellulose column chromatography. Their findings show that the parasite AST specific activity is less than mammalian tissues one (13). This difference between ours and these results could be related to some factors such as differences between used methods for sample preparing and enzyme s value detecting; also the difference between selected isolates of parasite. In our study, AST activity of treated parasite and untreated group is not significant. According to the study performed in Germany on the value of the liver enzymes in the serum of the Cuban patients infected with fasciola, the level of AST elevated from 3d to 7th day posttherapy with TCBZ and after that it decreased to normal 150

3 Table 1. The mean values of protein concentration and ASTs activity for F. gigantica parasite and liver samples. Extraction Samples Protein amounts (mg/ml) AST Total activity (U/mL)* AST Specific activity (U/mL/mg protein) Healthy liver Untreated Parasite Treated Parasite** Infected liver *One unit of enzyme activity is the quantity of enzyme that catalyzes the reaction of 1µmol of substrate (Aspartate) per minute at 37 C ** Treated parasite by Egaten (15μg/mL) Figure 1. SDS-PAGE analysis and molecular weight (kda) detection of proteins from the extracts of parasite samples of treated (by triclabendazole) and untreated. Lanes 2-5 are test group (treated). Lanes 6-9 are control samples (untreated). Lanes 1 is protein marker. Figure 2. SDS-PAGE analysis and molecular weight (kda) detection of proteins from the extracts of liver samples. Lanes 1 is healthy liver group. Lane 4 is infected liver. Lanes 5 is protein marker. 151

4 Table 2. Recognized proteins from Fasciola gigantica parasite by using expasy protein database. Protein Bands MW in Dalton protein name Not adapted expasy protein bank list Not adapted expasy protein bank list SAP Not adapted expasy protein bank list Not adapted expasy protein bank list Not adapted expasy protein bank list Not adapted expasy protein bank list Not adapted expasy protein bank list Not adapted expasy protein bank list Not adapted expasy protein bank list Not adapted expasy protein bank list NADH dehydrogenase Glucose transporter&glucose transporter-2 protein NADH dehydrogenase AST enzyme&nadh dehydrogenase Cytochrome b Cathepsin L& Cathepsin B NADH dehydrogenase NADH dehydrogenase Vitelline protein BIl Tegumental calcium-binding EF-hand protein&nadh-ubiquinone oxidoreductase chain Peroxiredoxin NADH-ubiquinone oxidoreductase chain NADH-ubiquinone oxidoreductase chain NADH-ubiquinone oxidoreductase chain NADH dehydrogenase value (14). Also in the other study performed in 2010 in Argentina for evaluation of resistance of fasciola against TCBZ in cattle, showed that these enzymes in 0 and 26th day post treatment with drug is decreased (15). In an effort in Cameroon in 2005, assaying biochemical parameters in control and treated goats showed no significant difference in serum level of AST and GGTs (ɤ-glutamyltranferase) (16). The results of our survey show decreasing in AST level of infected liver when to compare with healthy one is significant. From the presented data of study in Egypt in 2008, the level of AST and GGTs in serum of patients infected with fasciola far elevated (17). The other study in 1998 on water buffaloes with chronic fasciolosis is mentioned that six weeks post infection, the level of transaminases in plasma elevated and showed significant difference (18). More surveys on these enzymes level in different phases of diseases and more accurately investigation of pathogenicity of different species of parasite are needed. At now there is not record for AST detection and TCBZ drug on enzyme level in F. gigantica and this is first report on Iranian isolates of parasite. 152

5 Table 3. Recognized proteins from liver sample by using expasy protein database. Protein Bands MW in Dalton Protein Name Not adapted expasy protein bank list Not adapted expasy protein bank list Not adapted expasy protein bank list Glycogen phosphorylase, brain form Not adapted expasy protein bank list Not adapted expasy protein bank list Not adapted expasy protein bank list NADH-ubiquinone oxidoreductase chain Retinal dehydrogenase AST enzyme&corticosteroid-binding globulin Acyl-CoA desaturase Ornithine carbamoyltransferase& mitochondrial Cytochrome c oxidase Beta-carotene oxygenase Somatotropin Conclusion At now there is not record for AST detection and TCBZ drug on enzyme level in F. gigantica and this is first report on Iranian isolates of parasite. Although AST could not be concerned as an indicator for drug efficiency, however may be involved as a biomarker for biochemical comparison of parasite and host tissue. Acknowledgment The authors wish to thank the personnel of local abattoir (Soleymani, Tehran, Iran) for collecting of livers. Financial support for this project provided by Tehran University of Medical Sciences (Grant #22957). References 1. Haseeb AN, el-shazly AM, Arafa MA, Morsy AT. A review on fascioliasis in Egypt. J Egypt Soc Parasitol 2002; 32(1): Van den Enden E. Pharmacotherapy of helminthes infection. Expert Opin Pharmacother 2009;10(3): Robinson MW, Trudgett A, Hoey EM, Fairweather I. Triclabendazole resistant Fasciola hepatica: beta-tubulin and response to in vitro treatment with triclabendazole. Parasitology 2002;124 (3): Barrett J. Amino Acid Metabolism in Helminthes. Advances in Parasitology 1991;30: Aspartate Aminotransferase. Wikipedia website (2014). Available from URL:wiki/Aspartate transaminase. 6. Berger L, Wilson J, Wood P, Berger B. Methionine Regeneration and Aspartate Aminotransferase in Parasitic Protozoa. J Bacteriol 2001; 183(15): Hutchinson GW, Dawson K, Fitzgibbon CC, Martin PJ. Efficacy of an injectable combination anthelmintic (nitroxynil+clorsulon+ivermectin) against early immature Fasciola hepatica compared to triclabendazole combination flukicides given orally or topically to cattle. Vet Parasitol 2009;162(3-4): Maizels RM, Blaxter ML, Robertson BD, Selkirk ME. Parasite antigen and parasite genes: A laboratory manual for molecular parasitology.1st ed. Cambridge University Press; Pars Azmoon, Aspartate Transaminase kit; number ExPASy SIB Bioinformatics Resource Portal. SIB Website Available from URL: Standard Deviation Calculator. Easy Calculation Website Available from URL: Varghese A, Raina OK, Nagar G, et al. Development of cathepsin-l cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes. Vet Parasitol 2012; 183(3-4): Sun HP, Nyon SK, Hi SL, Chul YS. Aspartate and alanine aminotransferase in Fasciola hepatica. Korean J Parasitol 1983; 21 (1): Millán JC1, Mull R, Freise S, Richter J. The efficacy and tolerability of triclabendazole in Cuban patients with latent and chronic Fasciola hepatica infection. Am J Trop Med Hyg 2000; 63 (5-6): Olaechea F, Lovera V, Larroza M, Raffo F, Cabrera R. Resistance of Fasciola hepatica against triclabendazole in cattle in Patagonia (Argentina). Vet Parasitol 2011; 178 (3-4): Mbuh JV, Mbwaye J. Serological changes in goats experimentally infected with Fasciola gigantica in Buea sub-division of S.W.P. Cameroon. Vet Parasitol 2005; 131 (3-4): El-Shazly AM, Azab MS, El-Beshbishi SN, et al. Biochemical parameters in chronic fascioliasis. J Egypt Soc Parasitol 2008; 38 (3): Yang Q, Mao WH, Ferre I, Bayón JE, Mao XZ, González-Gallego J. Plasma aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transpeptidase (GGT) activities in water buffaloes with experimental subclinical fasciolosis. Vet Parasitol 1998; 78 (2):