Antidepressant and antipsychotic drugs differentially affect PON1 enzyme activity

Abstract

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 K_i of 4.15 and 0.007 mM, respectively.

• Antidepressant
• antipsychotic
• IC₅₀
• inhibition
• paraoxonase
• purification

Introduction

Paraoxonase (PON) (aryldialkyl phosphatase, E.C.3.1.8.1) is a calcium dependent serum esterase. The human paraoxonase (PON) gene family consists of three members, PON1, PON2 and PON31. The three PON genes show a high similarity at the amino acid level between the mammalian species PON1 and PON3, which are expressed primarily in the liver. In contrast, PON2 is widely expressed in a number of tissues including brain, liver, kidney and testis and it may have multiple mRNA forms2^,3.

PON1 is synthesized primarily in the liver and a portion is secreted into the plasma. PON1 is associated with high-density lipoproteins (HDL)4^,5. Therefore, PON1’s primary physiological role is to protect low-density lipoproteins. Purified PON1 has a molecular mass of 43–45 kDa and contains as many as three carbohydrate chains accounting for 15.8% of the weight6. Unlike PON2 and PON3, PON1 is an efficient esterase towards many OP (organophosphate) compounds including paraoxon, the insecticides parathion and chlorpyriphos as well as the nerve agents sarin and soman. PON1 received its name from paraoxon, the toxic metabolite of the insecticide parathion, which is one of its most studied substrates1^,7–9. In addition, PON1 was shown to hydrolyze over 30 lactones and cyclic carbonate esters including drugs and endogenous compounds5^,10–12.

In addition to its important roles in a broad range of fields, PON1 has been shown to play a critical role in the metabolism of pharmaceutical drugs2. PON1 activity is completely dependent upon calcium, and EDTA irreversibly abolishes its activity. Other cations, however, have been shown to have an inhibitory effect on PON1 activity. Barium, lanthanum, copper, zinc and mercurials were found to inhibit PON1 activity from rat or human liver13. Most studies on the modulation of PON1 by pharmaceutical compounds have focused on lipid-lowering compounds14. Since paraoxonase enzyme (PON1) is a bioscavenger against both atherosclerosis and organophosphate toxicity, studies on paraoxonase enzyme (PON) occupy an important place in the scientific world. Due to these vital factors, we believe that further studies on PON–drug interactions are needed. In our previous study, we reported that some drugs differentially affect the PON1 enzyme activity from human serum in vitro15–17. In this study, the effects of several types of antidepressant (Fluoxetine hydrochloride and Acepromazine) and antipsychotic (Haloperidol and Diazepam) drugs have been investigated on paraoxonase 1 enzyme activity purified from human blood (Figure 1). This study will be the first study on the effect of antidepressant and antipsychotic drugs on paraoxonase enzyme activities.

Figure 1. Molecular structure of antidepressant and antipsychotic drugs: [A] Fluoxoetine hydrochloride, [B] Haloperidol, [C] Acepromazine and [D] Diazepam.

Methods

Materials

Sepharose 4B, l-tyrosine, 1-napthylamine, protein assay reagents and chemicals for electrophoresis and all other chemicals used were of analytical grade and obtained from either Sigma (St. Louis, MO) or Merck (Darmstadt, Germany). Medical drugs were provided by the local pharmacy.

Purification of paraoxonase from human serum by hydrophobic interaction chromatography

Human serum was isolated from 35 mL fresh human blood and put into a dry tube. The blood samples were centrifuged at 1500  rpm for 15 min and the serum was removed. First, serum paraoxonase was isolated by ammonium sulfate precipitation (60–80%). The precipitate was collected by centrifugation at 15 000 rpm for 20 min, and redissolved in 100 mM Tris–HCl buffer (pH 8.0). Next, we synthesized the hydrophobic gel, including Sepharose 4B, l-tyrosine and 1-napthylamine, for the purification of human serum paraoxonase). The column was equilibrated with 0.1 M of a Na₂HPO₄ buffer (pH 8.00) including 1 M ammonium sulfate. The paraoxonase was eluted with an ammonium sulfate gradient using 0.1 M Na₂HPO₄ buffer with and without ammonium sulfate (pH 8.00). The purified PON1 enzyme was stored in the presence of 2  mM calcium chloride to maintain activity.

Total protein determination

The absorbance at 280 nm was used to monitor the protein in the column effluents and ammonium sulfate precipitation. Quantitative protein determination was achieved by absorbance measurements at 595  nm according to the Bradford method with bovine serum albumin as a standard.

SDS polyacrylamide gel electrophoresis

SDS polyacrilamide gel electrophoresis was performed after purification of the enzyme. It was carried out in 10 and 3% acrylamide concentration for the running and stacking gel, respectively, containing 0.1% SDS according to Laemmli18. A 20 mg sample was applied to the electrophoresis medium. Gel was stained overnight in 0.1% Coomassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid, then destained by frequently changing the same solvent, without dye. The electrophoretic pattern was photographed with the system of produce as an image of the gel.

Paraoxonase enzyme assay

Paraoxonase enzyme activity towards paraoxon was quantified spectrophotometrically by the method described by Gan et al.6. 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 determined in duplicate and corrected for the non-enzymatic hydrolysis.

In vitro inhibition kinetic studies and determination of K_i values

For the inhibition studies of Haloperidol, Fluoxetine hydrochloride, Diazepam and Acepromazine, different concentrations of each medical drugs were added to the enzyme activity. Paraoxonase activity with medical drugs was assayed by following the hydration of paraoxon. Activity % values of paraoxonase for five different concentrations of each medical drug were determined by regression analysis using Microsoft Office 2003 Excel. Paraoxonase activity without a medical drug was accepted as 100% activity. For the drugs having an inhibition effect, the inhibitor concentration causing up to 50% inhibition (IC₅₀ values) was determined from the graphs.

Results

To investigate the effect of these drugs on PON1 enzyme activity in vitro, human serum paraoxonase was purified by ammonium sulfate precipitation at 60–80% intervals, and subjected to hydrophobic interaction chromatography. The gel for hydrophobic interaction chromatography was synthesized using Sepharose 4B, l-tyrosine and 1-napthylamine. The overall purification of human serum PON1 was obtained in a activity of 109.29 U/mL and this enzyme was purified 125-fold (Table 1). The purity of the enzyme was confirmed by SDS-PAGE. As shown in Figure 2, a single band, 43 kDa, was obtained, which corresponds to the results of previous studies16. As shown in Figure 3, Fluoxetine hydrochloride and Haloperidol in vitro, inhibited the human serum PON1 activity as seen in Table 2 (Figure 3). However, Diazepam and Acepromazine did not show any inhibition effect on PON1 activity (Figure 3). In addition, the kinetics of the interaction of well-inhibited drugs, Fluoxetine hydrochloride and Haloperidol with the purified human serum PON1 was also studied. The Lineweaver–Burk double-reciprocal graph was plotted using a range of paraoxon concentrations (0.4–8.4 mM) in the absence or presence of each drug. KM and V_max values were determined by means of these graphs. The K_i value was calculated by the method of Dixon plots, which provides a simple way of determining the inhibition constant. The kinetic data indicate that the inhibition of paraoxonase activity by Fluoxetine hydrochloride and Haloperidol was of the uncompetitive type (Figure 3 and Table 2).

Figure 2. SDS-PAGE of PON1 purified by ammonium sulfate precipitation and hydrophobic interaction chromatography gel serum hPON1 was purified with ammonium sulfate precipitation (60–80%) and hydrophobic interaction chromatography. Lane 1, a pooled sample obtained from a column showing paraoxonase enzyme activity. Lane 2 contains β-galactosidase (116 kDa), bovine serum albumin (66.0 kDa), ovalbumin (45 kDa), Carbonic Anhydrase (33.0 kDa), β-lactogloulin (25.0 kDa), lysozyme (19.5 kDa) protein marker. The molecular weight of PON1 was estimated to be approximately 43 kDa.

Figure 3. Inhibition of paraoxonase hydrolysis by Acepromazine (A), Diazepam (B), Fluoxetine hydrochloride (C), and Haloperidol (D). A purified paraoxonase from human serum was assayed for paraoxonase activity at pH 8 in the presence of various concentrations of Haloperidol, Fluoxetine hydrochloride, Diazepam and Acepromazine. IC₅₀ values of these were determined from C and D. The slope of Lineweaver-Burk plots indicates uncompetitive for Fluoxetine hydrochloride (E) and Haloperidol (F).

Table 1. Summary of the purification of human serum Paraoxonase.

Step	Volume (mL)	Activity (U/mL)	Total activity (U)	Protein content (mg/mL)	Total protein (mg)	Specific activity (U/mg)	% Yield	Purification Fold
Serum	57	57.72	3290.04	1271.34	72 466.38	0.0454	100	–
Ammonium sulphate precipitation	20	85.96	1719.2	1084.8	21 696	0.0791	52.25	1.74
Hydrophobic interaction chromatography	3	109.29	327.87	10.98	32.94	9.95	9.96	125.6

Units: 1 nmol: 4-nitrophenol formed per minute. Purification (fold): specific activity, n purification step/specific activity in serum. Yield: activity of fractions combined for the next purification step/total activity in serum × 100. Yields figures do not include all of the activity actually recovered. Usually, three tubes were pooled for hydrophobic interaction chromatography.

Table 2. The effects of Fluoxetine hydrochloride and Haloperidol on the purified human serum PON1 and the inhibition type of drugs.

Drugs	IC50 (mM)	Inhibition type
Fluoxetine hydrochloride	3.08	Uncompetetive
Haloperidol	0.187	Uncompetetive

Discussion

Human serum paraoxonase (Arylesterase, EC 3.1.8.1, PON1) can hydrolyze organophosphate compounds and aryl esters, but its physiological substrates are still unknown19. In addition, paraoxonase is one of the most important enzymes in lipid metabolism, cardiovascular disease and atherosclerosis. PON1 activity also decreases in pathophysiologic conditions such as hypercholesterolemia, smoking habit, aging, obesity, menopause and acute renal failure20. For instance, excessive smokers were found to have high serum LDL and other lipid levels. Indeed, PON1 activity and HDL levels of drinkers were determined to be lower than non-drinkers. Along with other major and minor risk factors, this increases the risk of coronary artery disease21.

Toxicology, enzyme–drug interactions and some chemical interaction studies with various enzymes are vital and the number of these studies is increasing every day worldwide22–24. In general, drugs and toxic substances display their biological effects by interaction with specific enzymes.

Costa et al.25 reported that modulation of PON1 have involved pharmaceutical drugs, particularly lipid-lowering compounds such as statins and fibrates, as well as some other drugs26–30. A few studies have investigated the effects of some lipid-lowering agents on serum paraoxonase activity31–34. The activity of serum PON1 was found to be increased in coronary patients who took low-dose aspirin (acetyl-salicylic acid)35. However, administration of aspirin to healthy volunteers did not alter serum PON1 activity in another study. Aspirin also increased PON1 activity in HuH7 cells36. Various other pharmaceutical drugs have been investigated in different experimental protocols for their effects on PON1 activityand/or expression. The cholinergic muscarinic antagonist atropine was reported to inhibit human plasma paraoxonase. In our previous studies certain antibiotics, such as sodium ampicillin, ciprofloxacin and clindamycin sulphate, but notrifamycin, inhibited purified human PON137. When the same antibiotics were given in vivo to mice, however, increases or decreases of serum PON1 activities at different time-points were observed, with similar results observed in the liver. It has also been reported that mouse liver PON activity decreased with some contraceptives while the serum PON activity increased15^,38.

The effect of different pharmaceutical drugs on paraoxonase enzyme activity can be used to clarify PON1 status in the metabolism. Given the physiological importance of paraoxonase, the study of the effect of antidepressant and antipsychotic ciatric drugs on paraoxonase enzyme activity is an increasingly important issue for human health. Although there are a huge number of studies describing the critical role of PON1 in a variety of diseases specifically cardiovascular disease and atherosclerosis, there is no other information available about the effects of antidepressants and antiphysotic drugs on PON1 activity.

In our study, to investigate the effect of these drugs on PON1 enzyme activity in vitro, human serum paraoxonase was purified by ammonium sulfate precipitation at 60–80% intervals, and subjected to hydrophobic interaction chromatography. Different protocols are available for PON1 enzyme purification from serum and liver using three, four and seven steps29^,30. We previously reported a purification strategy designed for the human PON1 enzyme consisting of two-step procedures resulting in a shorter and more straightforward approach in contrast to other purification procedures29. Later, we investigated the in vitro effects of some antidepressant and antipsychotic drugs such as Fluoxetine hydrochloride (A), Haloperidol (B), Acepromazine (C) and Diazepam drugs (D) on PON1 activity. We found that compounds A and B inhibited enzyme activity non-competitively, compounds C and D did not show the inhibition efficiency. We can conclude from the table that Fluoxetine hydrochloride is the more efficient inhibitor than haloperidol. Fluoxetine inhibit many isozymes of the cytochrome P450 system that make drug metabolism possible. Both are potent inhibitors of CYP2D6 (the main enzyme responsible for their metabolism) and mild to moderate inhibitors of CYP1A2, CYP2B6, CYP2C9/2C19 and CYP3A4; furthermore, they inhibit the activity of P-glycoprotein, a type of membrane transport protein that plays an important role in drug transport and metabolism. This extensive effect on the body's pathways for drug metabolism creates the potential for interactions with many commonly used drugs39.

However, there are still few studies on PON enzyme activity–drug interaction. In the light of the above information, it could be argued that people cannot be thought to stay away from various chemicals including drugs for their health. However, it is vital to specifically adjust drug-dosages for person and disease. In this study, we purified human serum paraoxonase enzyme using three simple purification steps and investigated the in vitro effects of the mentioned antidepressant and antipsychotic drugs on enzyme activity.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Acknowledgements

This work was carried out in the Balikesir University Research Center of Applied Sciences (BURCAS).