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Hepg2) in Vitro See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/7315872 Amphenicol and macrolide derived antibiotics inhibit paraoxonase enzyme activity in human serum and human hepatoma cells (HepG2) in vitro Article in Biochemistry (Moscow) · February 2006 DOI: 10.1134/S0006297906010068 · Source: PubMed CITATIONS READS 17 85 5 authors, including: Selma Sinan Feray Kockar Balikesir University Balikesir University 36 PUBLICATIONS 435 CITATIONS 65 PUBLICATIONS 579 CITATIONS SEE PROFILE SEE PROFILE Hatice yıldırım Balikesir University 15 PUBLICATIONS 125 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Kanser Tedavisinde Kullanılabilecek, Moleküler Modelleme Tabanlı Yeni Nitroredüktaz (Ntr)/İlaç-Öncü (Prodrug) Sistemlerin Geliştirilmesi ve Enzim Aktivite-Sitotoksisite Araştırmaları View project magnetic nanoparticles View project All content following this page was uploaded by Feray Kockar on 19 May 2015. The user has requested enhancement of the downloaded file. ISSN 00062979, Biochemistry (Moscow), 2006, Vol. 71, No. 1, pp. 4650. © Pleiades Publishing, Inc., 2006. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 1, pp. 5964. Originally published in Biochemistry (Moscow) OnLine Papers in Press, as Manuscript BM05068, June 12, 2005. Amphenicol and Macrolide Derived Antibiotics Inhibit Paraoxonase Enzyme Activity in Human Serum and Human Hepatoma Cells (HepG2) in vitro S. Sinan1, F. Kockar1, N. Gencer2, H. Yildirim1, and O. Arslan2* 1Balikesir University, Science and Art Faculty, Department of Biology/Biochemistry Section, 10100 Balikesir, Turkey 2Balikesir University, Science and Art Faculty, Department of Chemistry/Biochemistry Section, 10100 Balikesir, Turkey; fax: 902662493360; Email: [email protected] Received March 10, 2005 Revision received March 23, 2005 Abstract—Human serum paraoxonase (PON1) was separately purified by ammonium sulfate precipitation and hydrophobic interaction chromatography. The in vitro effects of commonly used antibiotics, namely clarithromycin and chlorampheni col, on purified human serum paraoxonase enzyme activity (serum hPON1) and human hepatoma (HepG2) cell paraox onase enzyme activity (liver hPON1) were determined. Serum hPON1 and liver hPON1 were determined using paraoxon as a substrate and IC50 values of these drugs exhibiting inhibition effects were found from graphs of hydratase activity (%) by plotting concentration of the drugs. We determined that chloramphenicol and clarithromycin were effective inhibitors of serum hPON1. DOI: 10.1134/S0006297906010068 Key words: paraoxonase, inhibition, clarithromycin, chloramphenicol, in vitro, HepG2 Human serum paraoxonase (hPON1) (EC 3.1.8.1) is tral to the initiation and progression of atherosclerosis an antioxidant enzyme physically related to high density [14]. The antioxidant activity of HDL relates to its lipoprotein (HDL) and has roles in blocking oxidation of enzymes, primarily PON1, but also lecithin cholesterol low density lipoprotein (LDL) and in detoxification of acyl transferase [15], and these can prevent lipid peroxide organophosphates [13]. PON1 is a member of a multi accumulation on LDL both in vitro and in vivo [13, 16 gene family that includes at least two other genes in 18]. humans and mice [4]. Previous studies showed that Most studies on the modulation of PON1 by phar PON1 possesses hydrolytic activity with organophos maceutical compounds have focused on lipidlowering phates such as paraoxon, soman, sarin, and tabun [57]. compounds. In vitro exposure of HuH7 human hepatoma PON activity has been found in a variety of mammalian cells to provastatin, simvastatin, and fluvastatin caused a tissues, with liver and serum having the highest levels [8], 2550% decrease in PON1 activity in the culture medium and the source of serum PON is believed to be primarily and a similar decrease in PON1 mRNA; both effects were the liver [9]. The serum HDL concentration is inversely reversed by mevalonate [19]. In the same cells, fenofibric correlated with atherosclerosis risk [10]. The initial focus acid caused a 50 and 30% increase in PON1 activity and of attention was on the role of HDL in reversecholesterol PON1 mRNA content, respectively [19]. In another in transport. However, recent studies have suggested more vitro study on isolated lipoproteins, two oxidized metabo diverse mechanisms. HDL protects against oxidative lites of atorvastatin and a metabolite of gemfibrozil were modification of LDL [1113]. This is believed to be cen found to increase HDLassociated PON1 activity [20]. A study in rats indicated that fluvastatin reduced both plas ma and liver PON1 activity, while a lower dose was only Abbreviations: PON1) paraoxonase enzyme; serum hPON1) effective toward liver activity. Provastatin, on the other human serum paraoxonase enzyme; liver hPON1) human liver paraoxonase enzyme in HepG2 cell line; HDL) high density hand, was devoid of significant inhibition effects [21]. lipoproteins; LDL) low density lipoproteins; HepG2) human Studies in humans have provided similar contrasting hepatoma cell. results. An increase in serum PON1 activity was found in * To whom correspondence should be addressed. patients treated with simvastatin and other statins, gemfi 46 INHIBITION STUDIES ON PARAOXONASE ENZYME 47 brozil and fenofbirate [2225]. On the other hand, no Total protein determination. The absorbance at changes in serum PON1 activity were reported by other 280 nm was used to monitor the protein in the column studies in patients treated with ciprofibrate [26], bezofi effluents and ammonium sulfate precipitation. brate, and gemfibrozil [27]. The cholinergic muscarinic Quantitative protein determination was achieved by antagonist, atropine, was shown to inhibit human plasma absorbance measurements at 595 nm according to and pig liver PON1 in vitro [28]. In a cohort of aspirin Bradford [34], with bovine serum albumin (BSA) stan users, a significant increase of plasma PON1 activity and dard. concentration was reported [29]. In addition, the anti Purification of paraoxonase from human serum by inflammatory glucocorticoid dexamethasone caused an hydrophobic interaction chromatography. Human serum eightfold increase in PON1 mRNA in a mouse hepatoma was isolated from 35 ml fresh human blood taken to dry cell line (Hepa cells), as well as in mice in vivo [30]. tube. The blood samples were centrifuged at 1500g for Clarithromycin is a macrolide antibiotic that is wide 15 min and the serum was removed. First, serum paraox ly used for the treatment of a myriad of infections such as onase was isolated with ammonium sulfate precipitation those caused by Hemophilus influenzae, Mycobacterium (6080%). The precipitate was collected by centrifugation avium, and Helicobacter pylori. Clarithromycin is oxida at 15,000g for 20 min and redissolved in 100 mM Tris tively metabolized to 14(R)hydroxyclarithromycin or HCl buffer (pH 8.0). Then, we synthesized hydrophobic Ndemethylated to Ndesmethylclarithromycin and gel including Sepharose 4BLtyrosine and 1naphthy members of the CYP3A subfamily mediate these reac lamine for the purification of human serum paraoxonase tions [31]. Like erythromycin, clarithromycin is a potent in our laboratory [35]. The synthesized gel was placed in mechanismbased inhibitor of CYP3A [32]. a 10 cm column. Subsequently, the column was equili Chloramphenicol is an amphenicol antibiotic, which is brated with 0.1 M Na2HPO4 buffer (pH 8.0) containing effective on Rickotsiae, LymphogranulomaPsittaccosis 1 M ammonium sulfate. The A280 absorbance of the col group and Vibrio cholera, and it is especially effective on lected fractions was measured throughout equilibration. Salmonella typhi and Hemophilus. The antibiotic chlo The equilibration was carried out to reach 0.009 ramphenicol is an irreversible inhibitor, which forms a absorbance value. The 15 ml sample was loaded onto the covalent amide bond with a lysine amino acid residue at column. First, a salt gradient was carried out with 0.1 M the active site of the enzyme cytochrome P450 [33]. Na2HPO4 buffer (pH 8.0) with and without 1 M ammo Many antibiotics are used to deal with some tissues nium sulfate buffer in a gradient mixer. After the comple disorders but there are few studies of their effects on tion of salt gradient, elution was performed with 15 ml enzyme activities. To our knowledge the effects of some 0.1 M Na2HPO4 buffer (pH 8.0) containing 2 mM CaCl2. widely used antibiotics on serum and liver paraoxonase Fractions were collected into 1.5 ml tubes and the PON have not been investigated. The present study therefore activity and protein concentrations were determined for investigated in vitro the effects of chloramphenicol and each tube. The purified PON enzyme was stored in the clarithromycin on serum hPON1 and on liver hPON1 in presence of 2 mM CaCl2 in order to maintain activity. HepG2 cell line. SDSpolyacrylamide gel electrophoresis (SDS PAGE). SDSPAGE was performed after the purification of the enzyme. It was carried out in 10 and 3% acrylamide MATERIALS AND METHODS concentration for the running and the stacking gel, respectively, containing 0.1% SDS according to Laemmli Materials. Sepharose 4B, Ltyrosine, 1naphthy [36]. A 20µg sample was applied to the electrophoresis lamine, protein assay reagents, and chemicals for elec medium. Gels were stained for 1.5 h in 0.1% Coomassie trophoresis were obtained from Sigma (USA). All other Brilliant Blue R250 in 50% methanol and 10%
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