Effects of thymoquinone and the curcumin analog EF-24 on the activity of the enzyme paraoxonase-1 in human glioblastoma cells U87MG.

The spices and aromatic herbs were used not only in cooking to add flavour and smell to dishes but also for medicinal use. Nigella sativa, also called black cumin, is one of the species that contains an important bioactive component, thymoquinone (TQ), which has antioxidant, anti-inflammatory, antimicrobial, and antidiabetic effects. Curcuma longa, which also includes curcumin, has numerous anti-cancer properties. However, the bioavailability of curcumin is lower than that of its analogs. An analog of curcumin (EF-24), which has better bioavailability than curcumin, is capable of exerting a high anti-cancer effect. In our study, we determined the effects of PON1 enzyme activity on the proliferation and aggressiveness of glioblastoma cancer treated with TQ and EF-24 from lysates of the glioblastoma cell line U87MG. The results were determined as increased PON1 activity after treatment with TQ and EF-24 in the U87MG cell line (p < 0.0001).

Assessment of antioxidant, acetylcholinesterase, paraoxonase inhibition activities and phenolic content of Alchemilla lithophila.

In the present study, antioxidant activity and inhibition of acetylcholinesterase (AChE) and paraoxonase (hPON 1) of Alchemilla lithophila extracts were evaluated for the first time. Besides, there is no research on the contents of phenolic compounds except for fatty acids. In this context, phenolic compounds of A. lithophila were investigated by liquid chromatography/ mass spectrometry (LC-MS/MS). The methanol extract of the A. lithophila exhibited significant inhibition on the AChE (IC50 value for methanol extract 0.162 ± 0.25 mg /mL, R2:0.992). Besides, antioxidant activities of the A. lithophila extracts were examined using by the methods ABTS•+ and DPPH• free radical scavenging potentials, FRAP and CUPRAC metal-reducing activities. ABTS•+ and DPPH• scavenging activities were found for methanol extract at 70.67% and water extract at 75.38%, respectively. Also, FRAP and CUPRAC metal-reducing were determined for water extract 0.796 and hexane extract 1.570 as absorbance. According to LC-MS/MS analyses, the amounts of ellagic acid, catechin hydrate, gallic acid, fumaric acid, luteolin, quercetin, kaempferol, acetohydroxamic acid, caffeic acid, syringic acid, hydroxybenzoic acid and salicylic acid were determined by LC-MS/MS, respectively. As a consequence, this study will be a useful resource for determining bioactivity and phenolic compound profile for natural medicine research.

Myeloperoxidase-induced modification of HDL by isolevuglandins inhibits paraoxonase-1 activity.

Reduced activity of paraoxonase 1 (PON1), a high-density lipoprotein (HDL)-associated enzyme, has been implicated in the development of atherosclerosis. Post-translational modifications of PON1 may represent important mechanisms leading to reduced PON1 activity. Under atherosclerotic conditions, myeloperoxidase (MPO) is known to associate with HDL. MPO generates the oxidants hypochlorous acid and nitrogen dioxide, which can lead to post-translational modification of PON1, including tyrosine modifications that inhibit PON1 activity. Nitrogen dioxide also drives lipid peroxidation, leading to the formation of reactive lipid dicarbonyls such as malondialdehyde and isolevuglandins, which modify HDL and could inhibit PON1 activity. Because isolevuglandins are more reactive than malondialdehyde, we used in vitro models containing HDL, PON1, and MPO to test the hypothesis that IsoLG formation by MPO and its subsequent modification of HDL contributes to MPO-mediated reductions in PON1 activity. Incubation of MPO with HDL led to modification of HDL proteins, including PON1, by IsoLG. Incubation of HDL with IsoLG reduced PON1 lactonase and antiperoxidation activities. IsoLG modification of recombinant PON1 markedly inhibited its activity, while irreversible IsoLG modification of HDL before adding recombinant PON1 only slightly inhibited the ability of HDL to enhance the catalytic activity of recombinant PON1. Together, these studies support the notion that association of MPO with HDL leads to lower PON1 activity in part via IsoLG-mediated modification of PON1, so that IsoLG modification of PON1 could contribute to increased risk for atherosclerosis, and blocking this modification might prove beneficial to reduce atherosclerosis.

Gadolinium-based contrast agents: in vitro paraoxonase 1 inhibition, in silico studies.

Medications show their biological effects by interaction with enzymes, which have been known to play an essential role in the pathogenesis of many diseases. Inhibition or induction of drug metabolizing enzymes has an essential place in the drug design for many kinds of diseases including cardiovascular, neurological, metabolic, and cancer. The main goal of the current study is to contribute to this growing drug design field by observing PON1-drug interactions. In recent years, the safety of gadolinium-based contrast agents (GBCAs) used in magnetic resonance imaging (MRI) has discussed. In the present study, paraoxonase 1 (PON1) enzyme was purified from human serum by simple chromatographic methods with 4095.24 EU mg-1 protein specific activity. The inhibitory activities of gadoteric acid, gadopentetic acid, gadoxetate disodium, and gadodiamide were investigated on PON1 activity of the enzyme. IC50 values were found in the range of 51.28 ± 0.14 to 285.80 ± 0.96 mM. Ki constants were found as 67.95 ± 0.60 mM, 104.97 ± 0.96 mM, 202.33 ± 1.75 mM, and 299.43 ± 2.64 mM for gadoteric acid, gadopentetic acid, gadoxetate disodium, and gadodiamide, respectively. While the inhibition types are determined as competitive of gadoxetate disodium and gadodiamide by the Lineweaver-Burk curves, it was noncompetitive for other compounds. In addition, the molecular docking analyses of gadoxetate disodium and gadodiamide were carried out to understand the binding interactions on the active site of the PON1 enzyme. The structure-activity relationship (SAR) of the drugs was established on the basis of different substituents and their positions in the compounds.

Evaluation of the paraoxonase-1 kinetic parameters of the lactonase activity by nonlinear fit of progress curves.

Although paraoxonase-1 (PON1) activity has been demonstrated to be a reliable biomarker of various diseases, clinical studies have been based only on relative comparison of specific enzyme activities, which capture differences mainly due to (usually unknown) PON1 concentration. Hence, the aim of this report is to present for the first time the simple evaluation method for determining autonomous kinetic parameter of PON1 that could be also associated with polymorphic forms and diseases; i.e. the Michaelis constant which is enzyme concentration independent quantity. This alternative approach significantly reduces the number of experiments needed, and it yields the results with great accuracy.

Naphthoquinones, benzoquinones, and anthraquinones: Molecular docking, ADME and inhibition studies on human serum paraoxonase-1 associated with cardiovascular diseases.

Paraoxonase-1 (PON1) has essential roles such as protecting low-density lipoprotein against detoxification and oxidation of highly toxic compounds. Quinones are a class of compounds and a type of plant-derived secondary metabolites. Here, PON1 was purified using very simple methods and evaluation of the interactions between the enzyme and some quinones. It was found that these quinones displayed effective inhibitor properties for PON1 with the IC50 values in the range of 3.27-82.90 µM and the K i values in the range of 2.50 ± 0.65 to 30.90 ± 7.20 µM. These quinones displayed distinct inhibition mechanisms. It was determined that except for 5-hydroxy-2-methyl-1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone all quinones exhibit competitive inhibition effects. Also, molecular docking and in silico ADME studies were performed. Usage of drugs including quinone derivatives in structure with biological activity would be hazardous in some cases.

Association of human serum paraoxonase-1 with some respiratory drugs.

In this study, we investigated the effects of certain respiratory drugs, which are mainly used on human serum paraoxonase-1 (hPON1; EC 3.1.8.1). hPON1 was purified from human serum, with 354.91 fold and 45% yield by using two simple step procedures including, first, ammonium sulfate precipitation, then, Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis showed a single protein band belonging to hPON1 with 43 kDa. All the pharmaceutical compounds inhibited the PON1 enzyme highly at the micromolar level. The obtained IC50 values for nine different pharmaceutics ranged from 0.219 µM (salbutamol sulfate) to 67.205 µM (montelukast sodium). So, all drugs could be considered as potent hPON1 inhibitors. Ki values and inhibition types were determined by Lineweaver-Burk graphs. While varenicline tartrate and moxifloxacin hydrochloride inhibited the enzyme in a noncompetitive manner, others inhibited it in a mixed manner.

Synthesis, characterization, and in vitro effect of the Cu(II) complex with niflumic acid and 3-picoline on paraoxanase-I.

Niflumic acid is used to treat inflammatory rheumatoid diseases, pain, and fever. The present study reports the experimental, spectroscopic, thermal, structural analyses, and biological activities of this complex. The nonsteroidal anti-inflammatory drug niflumic acid, 3-picoline, and copper(II) chloride were utilized to synthesize a new complex: [Cu2 Cl 2 (nif) 2 (3-pic) 4 ]. The crystal structure of [Cu 2 Cl 2 (nif) 2 (3-pic) 4 ] was determined by X-ray crystallography. The complex crystallizes in the triclinic space group P-1 and each Cu(II) center displayed six-coordinated distorted octahedral geometry. Two Cu(II) centers are connected by a chloro-bridge to form the binuclear metal core. Finally, the in vitro effects of the synthesized new complex and free niflumic acid were evaluated on the human serum paraoxonase 1 enzyme. At low doses, both the new complex and free niflumic acid showed very good inhibition activity with different inhibition mechanisms. In addition, the results showed that the new complex has more inhibition activity than free niflumic acid.

Synthesis and characterization of four novel palladium(II) and platinum(II) complexes with 1-(2-aminoethyl)pyrrolidine, diclofenac and mefenamic acid: In vitro effect of these complexes on human serum paraoxanase1 activity.

In this study, the effects of four novel mononuclear palladium(II) and platinum(II) complexes on the activity of human serum paraoxanase1 were examined. First, four novel mononuclear palladium(II) and platinum(II) complexes were synthesized with a nitrogen donor ligand 1-(2-aminoethyl)pyrrolidine and nonsteroidal anti-inflammatory drugs diclofenac, mefenamic acid. These complexes were characterized by spectroscopic, thermal, and elemental analyses. The crystal structures of complex [Pd(2-amepyr)2 ](dicl)2 1 and [Pd(2-amepyr)2 ](mef)2 3 were determined by X-ray crystallography. Then, paraoxonase1 enzyme was purified from human serum. The effects of these complexes on enzyme were evaluated in vitro. The complexes consist of the cationic unit and the counterions. The diclofenac and mefenamic acid acted as a counterion in the complexes. It was observed that all the complexes were stable up to high temperatures. These complexes, even at low doses, inhibited the activity of the enzyme with different inhibition mechanisms.

Assessment of the inhibitory effects and molecular docking of some sulfonamides on human serum paraoxonase 1.

Paraoxonase-1 (PON1) is an organophosphate hydrolyzer and antiatherogenic enzyme. Due to the PON1's crucial functions, inhibitors and activators of PON1 must be known for pharmacological applications. In this study, we investigated the in vitro effects of some sulfonamides compounds on human serum PON1 (hPON1). For this aim, we purified the hPON1 from human serum with high specific activity by using simple chromatographic methods, and after the purification processes, we investigated in vitro interactions between the enzyme and some sulfonamides (2-amino-5-methyl-1,3-benzenedisulfonamide, 2-chloro-4-sülfamoilaniline, 4-amino-3-methylbenzenesulfanilamide, sulfisoxazole, sulfisomidine, and 5-amino-2-methylbenzenesulfonamide). IC50 , Ki values, and inhibition types were calculated for each sulfonamide. 2-amino-5-methyl-1,3-benzenedisulfonamide and 2-chloro-4-sülfamoilaniline exhibited noncompetitive inhibition effect, whereas 4-amino-3-methylbenzenesulfanilamide, sulfisoxazole, and sulfisomidine exhibited mixed type inhibition. On the other hand, 5-amino-2-methylbenzenesulfonamide showed competitive inhibition and so molecular docking studies were performed for this compound in order to assess the probable binding mechanism into the active site of hPON1.

Antiepileptic drugs: Impacts on human serum paraoxonase-1.

Serum paraoxonase (PON1) is a key enzyme related to high-density lipoprotein (HDL)-cholesterol particle. It can prevent the oxidation of low-density lipoprotein (LDL) and HDL. The present article focuses on the in vitro inhibition role of some antiepileptic drugs (AEDs) such as valproic acid, gabapentin, primidone, phenytoin, and levetiracetam on human paraoxonase (hPON1). Therefore, PON1 was purified from human serum with a specific activity of 3976.36 EU/mg and 13.96% yield by using simple chromatographic methods. The AEDs were tested at various concentrations, which showed reduced in vitro hPON1 activity. IC50 values for gabapentin, valproic acid, primidone, phenytoin, and levetiracetam were found to be 0.35, 0.67, 0.87, 6.3, and 53.3 mM, respectively. Ki constants were 0.261 ± 0.027, 0.338 ± 0.313, 0.410 ± 0.184, 10.3 ± 0.001, and 43.01 ± 0.003 mM, respectively. Gabapentin exhibited effective inhibitory activity as compared with the other drugs. The inhibition mechanisms of all compounds were noncompetitive.

Functionalized imidazolium and benzimidazolium salts as paraoxonase 1 inhibitors: Synthesis, characterization and molecular docking studies.

Paraoxonase (PON) is a key enzyme in metabolism of living organisms and decreased activity of PON1 was acknowledged as a risk for atherosclerosis and organophosphate toxicity. The present study describes the synthesis, characterization, PON1 inhibitory properties and molecular docking studies of functionalized imidazolium and benzimidazolium salts (1a-5g). The structures of all compounds were elucidated by IR, NMR, elemental analysis and structures of compounds 2b and 2c were characterized by single-crystal X-ray diffraction. Compound 1c, a coumarin substituted imidazolium salt showed the best inhibitory effect on the activity of PON1 with good IC50 value (6.37 µM). Kinetic investigation was evaluated for this compound and results showed that this compound is competitive inhibitor of PON1 with Ki value of 2.39 µM. Molecular docking studies were also performed for most active compound 1c and one of least active compound 2c in order to determine the probable binding model into active site of PON1 and validation of the experimental results.

Some coumarins and benzoxazinones as potent paraoxonase 1 inhibitors.

In this study, we aimed to investigate the effect of some coumarin and benzoxazinone derivatives on the activity of human PON1. Human serum paraoxonase 1 was purified from fresh human serum blood by two-step procedures that are ammonium sulfate precipitation (60-80%) and then hydrophobic interaction chromatography (Sepharose 4B, L-tyrosine and 1-napthylamine). The enzyme was purified 232-fold with a final specific activity of 27.1 U/mg. In vitro effects of some previously synthesized ionic coumarin or benzoxazinone derivatives (1-21) on purified PON1 activity were investigated. Compound 14 (1-(2,3,4,5,6)-pentamethylbenzyl-3-(6,8-dimethyl-2H-chromen-2-one-4-yl))benzimidazolium chloride was found out as the strongest inhibitor (IC50 = 7.84 µM) for PON1 among the compounds. Kinetic investigation and molecular docking study were evaluated for one of the most active compounds (compound 12) and obtained data showed that this compound is competitive inhibitor of PON1 and interact with Leu262 and Ser263 in the active site of PON1. Moreover, coumarin derivatives were found out as the more potent inhibitors for PON1 than benzoxazinone derivatives.

In vitro inhibition effect of some coumarin compounds on purified human serum paraoxonase 1 (PON1).

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro effect of some hydroxy and dihydroxy ionic coumarin derivatives (1-20) on purified PON1 activity was investigated. Among these compounds, derivatives 11-20 are water soluble. In investigated compounds, compounds 6 and 13 were found the most active (IC50 = 35 and 34 µM) for PON1, respectively. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.

An alternative purification method for human serum paraoxonase 1 and its interactions with anabolic compounds.

In this study, an alternative purification method for human paraoxonase 1 (hPON1) enzyme was developed using two-step procedures, namely, ammonium sulfate precipitation and Sepharose-4B-L-tyrosine-3-aminophenantrene hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent M(W) of 43 kDa. The enzyme was purified 219-fold with a final specific activity of 4,408,400 U/mg and a yield of 10%. Furthermore, we examined the in vitro effects of some anabolic compounds, such as zeranol, 17 ß-estradiol, diethylstilbestrol, oxytocin, and trenbolone on the enzyme activity to understand the better inhibitory properties of these molecules. The five anabolic compounds dose dependently decreased the activity of hPON1 with inhibition constants in the millimolar-micromolar range. The results show that these compounds exhibit inhibitory effects on hPON1 at low concentrations with IC50 values ranging from 0.064 to 16.900 µM.

Toxic effects of methamidophos on paraoxonase 1 activity and on rat kidney and liver and ameliorating effects of alpha-tocopherol.

The role of alpha-tocopherol on nephrotoxicity and hepatotoxicity induced by methamidophos (MT) was investigated in wistar rats. Animals were given via gavage, for four weeks, a low dose of MT (MT1), a high dose of MT (MT2), vitamin E (200 mg/kg of bw) or both MT2 plus vitamin E (Vit E) and control group was given distillate water. MT treatment resulted in a significant decrease in the body weight of MT2-treated group. Moreover, MT-treated groups had significantly lower butyrylcholinesterase (p < 0.01) and paraoxonase 1 (PON1) activities compared with the control group (p < 0.05). However, MT2-treated group had significantly higher alkaline phosphatase activity compared with untreated rats (p < 0.05). Both MT-treated groups had significantly higher urea (p < 0.01) and uric acid levels (p < 0.05) compared with the control group. However, significant low uric acid level (p < 0.05) was noted in MT2 plus vit E-treated rats compared with MT2-treated group. Histopathological changes in organ tissues were observed in both MT-treated groups and MT2 plus vit E-treated rats. However, the damage was reduced in MT2 plus vit E-treated rats. Therefore, this study deduces that alpha-tocopherol administration may ameliorate the adverse effects of subacute exposure to MT on rat liver and kidney and this antioxidant can protect PON1 from oxidative stress induced by this organophosphorus pesticide. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 842-854, 2016.

Paraoxonase 1 activity in subchronic low-level inorganic arsenic exposure through drinking water.

Epidemiological evidences indicate close association between inorganic arsenic exposure via drinking water and cardiovascular diseases. While the exact mechanism of this arsenic-mediated increase in cardiovascular risk factors remains enigmatic, epidemiological studies indicate a role for paraoxonase 1 (PON1) in cardiovascular diseases. To investigate the association between inorganic arsenic exposure and cardiovascular diseases, rats were exposed to sodium arsenite (trivalent; 50, 100, and 150 ppm As) and sodium arsenate (pentavalent; 100, 150, and 200 ppm As) in their drinking water for 12 weeks. PON1 activity towards paraoxon (PONase) and phenylacetate (AREase) in plasma, lipoproteins, hepatic, and brain microsomal fractions were determined. Inhibition of PONase and AREase in plasma and HDL characterized the effects of the two arsenicals. While the trivalent arsenite inhibited PONase by 33% (plasma) and 46% (HDL), respectively, the pentavalent arsenate inhibited the enzyme by 41 and 34%, respectively. AREase activity was inhibited by 52 and 48% by arsenite, whereas the inhibition amounted to 72 and 67%, respectively by arsenate. The pattern of inhibition in plasma and HDL indicates that arsenite induced a dose-dependent inhibition of PONase whereas arsenate induced a dose-dependent inhibition of AREase. In the VLDL + LDL, arsenate inhibited PONase and AREase while arsenite inhibited PONase. In the hepatic and brain microsomal fractions, only the PONase enzyme was inhibited by the two arsenicals. The inhibition was more pronounced in the hepatic microsomes where a 70% inhibition was observed at the highest dose of pentavalent arsenic. Microsomal cholesterol was increased by the two arsenicals resulting in increased cholesterol/phospholipid ratios. Our findings indicate that decreased PON1 activity observed in arsenic exposure may be an incipient biochemical event in the cardiovascular effects of arsenic. Modulation of PON1 activity by arsenic may also be mediated through changes in membrane fluidity brought about by changes in the concentration of cholesterol in the microsomes.

Human serum paraoxonase-1 (hPON1): in vitro inhibition effects of moxifloxacin hydrochloride, levofloxacin hemihidrate, cefepime hydrochloride, cefotaxime sodium and ceftizoxime sodium.

In this study, we investigated the effects of antibacterial drugs (moxifloxacin hydrochloride, levofloxacin hemihidrate, cefepime hydrochloride, cefotaxime sodium and ceftizoxime sodium) on human serum paraoxonase-1 (hPON1) enzyme activity from human serum in vitro conditions. For this purpose, hPON1 enzyme was purified from human serum using simple chromatographic methods. The antibacterial drugs exhibited inhibitory effects on hPON1 at low concentrations. Ki constants were calculated to be 2.641 ± 0.040 mM, 5.525 ± 0.817 mM, 35.092 ± 1.093 mM, 252.762 ± 5.749 mM and 499.244 ± 10.149 mM, respectively. The inhibition mechanism of moxifloxacin hydrochloride was competitive, whereas levofloxacin hemihidrate, cefepime hydrochloride, cefotaxime sodium and ceftizoxime sodium were noncompetitive inhibitors.

Antidepressant and antipsychotic drugs differentially affect PON1 enzyme activity.

Human serum paraoxonase (PON1, EC 3.1.8.1.) is a high-density lipid (HDL)-associated, calcium-dependent enzyme. In this study, the effects of Haloperidol, Fluoxetine hydrochloride, Diazepam and Acepromazine drugs used for the therapy of antidepressant and antipsychotic diseases, on paraoxonase enzyme activity was studied in in vitro inhibition studies on purified human serum PON1. PON1 enzyme was purified from human blood using two-step procedures, namely, ammonium sulfate precipitation and sepharose-4B-l-tyrosine-1-napthylamine hydrophobic interaction chromatography. The overall purification of human serum PON1 was obtained in a activity of 109.29 U/mL and this enzyme was purified 125-fold. The SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Inhibition studies indicated that haloperidol and fluoxetine hydrocloride were effective inhibitors on purified human serum PON1 activity with IC50 of 0.187 and 3.08 mM values, respectively. The kinetics of interaction of haloperidol and fluoxetine hydrocloride with the purified human serum PON1 indicated uncompetitive inhibiton pattern with Ki of 4.15 and 0.007 mM, respectively.

Paraoxonase activity as a marker of exposure to xenobiotics in tobacco smoke.

The paraoxonase (PON) family is composed of 3 proteins (PON1, PON2, and PON3), each of which plays a crucial role in the body, displaying antioxidant, anti-inflammatory, and antiatherosclerotic properties. The activities and properties of PON proteins can be modulated by a number of environmental factors, including cigarette smoke. In the present article, a review of existing literature is employed to analyze both the direct and the indirect impact of cigarette smoking on the activity of members of the PON family. Cigarette smoking leads to direct inhibition of the hydrolytic activity of PON enzymes by modification of thiol groups, by the reactions of free radicals, or by inhibiting enzyme-active regions with heavy metals. It has been shown that cigarette smoking correlates with a decrease in high-density lipoprotein (HDL) concentration as well as with an increase in other components of the lipid profile (low-density lipoprotein (LDL), triglycerides, and total cholesterol). By decreasing HDL levels, cigarette smoking likely acts indirectly to induce a decline in PON1 activity. Inhibition of PON1 activity by smoking is a reversible process after cessation of exposure to the xenobiotics in tobacco smoke.