Effects of Some Antibiotics on Paraoxonase from Human Serum in Vitro and from Mouse Serum and Liver in Vivo

Selma Sinan, Feray Kockar, Nahit Gencer, Hatice Yildirim, Oktay Arslan
2006 Biological and Pharmaceutical Bulletin  
Serum paraoxonase (aryldialkilphosphatase, EC 3.1.8.1., PON1) is an esterase protein synthesized by the liver 1) and released into the serum, where it is associated with HDL (high density lipoprotein). The enzyme derives its name from its ability to hydrolyze paraoxon into p-nitrophenol and diethyl phosphate. 2) Paraoxon, a potent acethylcolinesterase inhibitor, is metabolically generated in vivo from the insecticide parathion by mitochondrial oxidation involving the cytochrome-P450 pathway. 3)
more » ... me-P450 pathway. 3) The ability to hydrolyze paraoxon is routinely used for measuring PON1 activity in vitro in serum samples. The enzyme catalyses the hydrolysis of a broad range of substrates including arylesters 1) and carbamates 4) as well as cyclic carbonates and lactones. Also, it hydrolyzes OP (organophosphate compounds). 5) Furthermore, the enzyme inhibits atherogenesis by preventing the oxidation of HDL and low-density lipoprotein (LDL). 6) PON1 also hydrolyzes homocysteine thiolactone and prevents protein homocysteinylation, the process involved in atherogenesis. 7) PON1 and closely related proteins, PON2 and PON3, have been identified. PON3 is also contained in HDL particles, 8, 9) whereas PON2 is not found in plasma but is expressed in many tissues. 10) PON2 and PON3 have antioxidant properties, but unlike PON1, lack paraoxon-hydrolyzing activity. More recently, in addition to its role in lipid metabolism, and hence in treating cardiovascular disease and arteriosclerosis, PON1 has been shown to play a role in the metabolism of pharmaceutical drugs. Given the physiological importance of paraoxonase, its metabolic impact on medically important drugs should receive greater study. However, there are not many inhibition studies available on paraoxonase activity. The inhibitory effects of some diuretic and hypocholes-terolemic drugs, such as spironolactone, mevastatin, lovastatin and simvastatin, pravastatin and prulifloxacin, have been investigated on terms of paraoxonase activity from human serum in vitro and in vivo. Differential effects of drugs on the PON enzyme activity was found. Some increased the activity and others decreased it. [11] [12] [13] Many antibiotics are being used in therapies. There are few reports related to changes in enzyme activities. To our knowledge, the effects of any antibiotics on serum or liver paraoxonase have not been investigated. Therefore, the aim of this study is to determine the effect of some antibiotics, namely sodium ampicillin, ciprofloxacin, rifamycin SV and clindamiycin phosphate, on purified human serum paraoxonase in vitro, and mouse serum and liver paraoxonase in vivo. MATERIALS AND METHODS Materials Sepharose 4B, L-tyrosine, 1-napthylamine, protein assay reagents and chemicals for electrophoresis were obtained from Sigma Chem. Co. All other chemicals used were of analytical grade and obtained from either Sigma or Merck. Medical drugs were provided by the local pharmacy. Paraoxonase Enzyme Assay Paraoxonase enzyme activity towards paraoxon was quantified spectrophotometrically by the method described by Gan et al. 14) The reaction was monitored for 2 min at 37°C by monitoring the appearance of p-nitrophenol at 412 nm in a Biotek automated recording spectrophotometer. The final substrate concentration during enzyme assay was 2 mM, and all rates were deter- August 2006August 1559 Paraoxonase (PON1, EC 3.1.8.1) is an esterase protein which plays multifunctional role in metabolism. Therefore, in this study the effects of commonly used antibiotics, namely sodium ampicillin, ciprofloxacin, rifamycin SV and clindamycin phosphate, on human PON1 were investigated in vitro and in vivo. Human serum paraoxonase (PON1) was separately purified by ammonium sulfate precipitation and hydrophobic interaction chromatography. The in vitro effects of the antibiotics in purifying human serum paraoxonase was determined using paraoxon as a substrate, and the IC 50 values of these drugs exhibiting inhibition effects were found from graphs of hydratase activity % by plotting the concentration of the drugs. It was determined that sodium ampicillin, ciprofloxacin, and clindamycin phosphate were effective inhibitors on human serum PON1, and the inhibition kinetics of interaction of sodium ampicillin, ciprofloxacin, and clindamycin phosphate with the human serum PON1 was also determined, with the K i of sodium ampicillin, ciprofloxacin, and clindamycin phosphate being 0.00714؎0.00068, 6.5؋10 ؊6 ؎4.59؋10 ؊7 , 0.0291؎0.0077 mM, respectively. The in vivo effects of the antibiotics on paraoxonase enzyme activity in mouse serum and liver PON1 were also investigated. Mouse liver PON1 activity showed a statistically significant change at 2, 4 and 6 h of drug appliciation in vivo. Sodium ampicillin and clindamycin phosphate exhibited about 80% mouse liver PON1 at 2 or 4 h ( p: 0.034, 0.003 and 0.021, respectively). In addition, ciprofloxacin and rifamycin SV only showed inhibition at 4 h incubation. Sodium ampicillin (17.12 mg/kg) lead to a significant decrease in mouse serum PON1 after 4 h drug administration. Ciprofloxacin (3.2 mg/kg), rifamycin SV (3.56 mg/kg) and clindamycin phosphate (2.143 mg/kg) did not exhibit any inhibition effect for the mouse serum PON1, in vivo.
doi:10.1248/bpb.29.1559 pmid:16880604 fatcat:3nepucma55glnkqeadt6zj5z5m