Identification of a new stilbene-derived inducer of paraoxonase 1 and ligand of the Aryl hydrocarbon Receptor

doi:10.1016/j.bcp.2011.12.013

Biochemical Pharmacology

Volume 83, Issue 5, 1 March 2012, Pages 627-632

https://doi.org/10.1016/j.bcp.2011.12.013 Get rights and content

Abstract

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme, synthesized in the liver and secreted into the blood. PON1 displays antioxidant properties and is involved in organophosphorous compounds and oxidized lipids degradation. Because of these beneficial effects, pharmacological regulation of PON1 appears to be highly relevant in toxicology and cardiology. Recent studies undertaken on the regulation of the PON1 promoter in our laboratory have identified resveratrol, through its activation of the Aryl hydrocarbon Receptor (AhR), as a putative inducer of PON1. We have tested a new modulator of AhR, (Z)-2,3-bis (4-nitrophenyl)-acrylonitrile, and established that it is a more potent inducer of PON1 at the mRNA, protein and enzymatic activity as compared to resveratrol. It also acts by activating the AhR. However, in contrast with traditional AhR agonists, it does not induce cyp1A1 transcription. (Z)-2,3-bis (4-nitrophenyl)-acrylonitrile is therefore a specific AhR modulator targeting PON1.

Graphical abstract

Introduction

Paraoxonase 1 (PON1) is an HDL-associated enzyme, which is synthesized in the liver. It was first characterized by its ability to hydrolyze organophosphorous (OP) compounds such as the insecticide paraoxon but it also hydrolyzes chemical warfare nerve agents such as soman. The enzyme has also been shown to be protective in the case of OP pesticides exposure [1]. More recently, a cardiovascular protective role has been suggested [2]. The latter role has been associated with different enzymatic properties of PON1: degradation of oxidized lipids [3], hydrolysis of homocystein thiolactone [4], and decreased cholesterol biosynthesis in macrophages [5]. Several polymorphisms of the PON1 gene have been identified in humans. These polymorphisms contribute to important inter-individual variations of PON1 [6]. In particular, polymorphisms located in the PON1 promoter are positively associated with higher concentrations of PON1 in the serum and with an increased risk of arterial ischemic stroke [7].

In light of the potentially protective role of PON1, the regulation of its gene has been extensively studied. Several factors, including commercial drugs, have been shown to regulate the level of PON1 [8]. For example, the exposure of human HuH7 cells to bezafibrate or fenofibric acid increases the level of PON1 mRNA as well as PON1 activity. The effect of statins on the regulation of PON1 (pravastatin, simvastatin and fluvastatin) is controversial. Indeed, two studies described increased levels of PON1 in serums of patients treated with simvastatin or atorvastatin [9], [10]. However, simvastatin did not exhibit any potential of increasing PON1 in HuH7 cells [11]. Other molecules with potential cardiovascular protective effects have been evaluated for their effects on PON1. For example, several natural polyphenols including quercetin and resveratrol stimulate PON1 activity. We have shown that the induction of paraoxonase 1 by polyphenols requires the transcription factor AhR (Aryl hydrocarbon Receptor). The AhR is a xenobiotic-activated receptor and a transcriptional regulator of enzymes involved in the metabolism of xenobiotics. The AhR forms a cytosolic complex with several heat shock proteins. Upon ligand binding, AhR translocates into the nucleus, and, following dissociation of the heat shock proteins, it binds to the nuclear protein ARNT (AhR Nuclear Translocator). The AhR/ARNT heterodimer binds to Xenobiotic Responsive Elements (XRE) located in the promoters of target genes [12]. Interestingly, a variant XRE has been identified in the promoter of PON1 [13]. Other studies have shown a link between the activation of the AhR and the induction of PON1. For example, aspirin increases PON1 expression in several cellular and animal models (HepG2, Sprague-Dawley rat hepatocytes, C57BL6 mice) but fails to induce PON1 in AhR−/− mice [14].

In order to identify a more potent inducer of PON1 than resveratrol, we designed several resveratrol-derived compounds as a continuing effort to develop AhR selective modulators. We describe here the properties of (Z)-2,3-bis (4-nitrophenyl)-acrylonitrile as a more potent inducer of PON1 as compared to resveratrol in the human hepatic cell line HuH7. Further, we show that this derivative activates the AhR.

Section snippets

Chemicals

TCDD was purchased from LCG Promochem (Molsheim, France). Benzo(a)pyrene, 3MC and quercetin were purchased from Sigma–Aldrich (St Louis, USA). Resveratrol was purchased from Cayman chemical (Ann Harbor, USA). (Z)-2,3-bis (4-nitrophenyl)-acrylonitrile (ZNPA) synthesized will be described elsewhere.

Cell culture

Human hepatocarcinoma HuH7 cells were maintained in Dulbecco's minimal essential medium (DMEM, Invitrogen, Cergy-Pontoise, France) supplemented with nonessential amino acids and containing 10% fetal

Results

Human hepatic HuH7 cells are frequently used to study PON1 regulation (e.g. [17], [18]). As a first step, we used a stably transfected HuH7 cell line previously established in our laboratory [11], which bears a PON1 promoter controlling the Firefly luciferase gene (Fig. 1a). This cell line was exposed to several resveratrol derivatives and luciferase expression was measured 48 h later, similarly to our former studies [19]. Resveratrol has been chosen for different reasons. First, its chemical

Discussion

Resveratrol displays important pharmacological properties such as its cardio-protective, anti-ageing and metabolic effects. Although it was initially believed that these effects were predominantely related to its antioxidant properties, it appears that this compound can also act through alternative mechanisms to modulate gene expression. Most of these effects have been observed in vitro within a wide range of concentrations [20]. Among its numerous effects, it has been demonstrated that

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The duration of treatments generally ranged from 24 to 48 h, while the doses of resveratrol ranged from 2 to 25 μM, with the exception of one study that used also concentrations of 200 μM [48]. Gouédard et al. [44] and Guyot et al. [50] reported an increased PON1 gene expression in human hepatocyte primary cultures and in HuH7 hepatoma cell line following a 48-h supplementation with 10 μM of resveratrol. Similar results were also observed by Gupta and colleagues [51] following an incubation of HepG2 cells for 48 h with 15 μM of resveratrol.
Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme involved in the protection of low-density lipoprotein and HDLs against lipid peroxidation. Several studies documented the capacity of polyphenols to stimulate PON1 transcription activation.
The objective of the present review is to provide the main evidence about the role and the potential mechanism of action of polyphenols and polyphenol-rich foods in the modulation of PON1 gene expression and activity.
A total of 76 in vitro and in vivo studies were included in the review. Overall, while evidence obtained in vitro is limited to quercetin and resveratrol, those deriving from animal models seem more convincing for a wide range of polyphenols but only at pharmacological doses. Evidence from human studies is promising but deserves more substantiation about the role of polyphenol-rich foods in the regulation of PON1 activity and expression.
Research focused on the understanding of the structure–activity relationship of polyphenols with PON1 and on the mechanisms at the base of PON1 modulation is warranted. Well-designed human intervention studies are encouraged to corroborate the findings of polyphenols also at physiological doses.
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Natural polyphenol resveratrol (trihydroxystilbene) is a partial agonist of human aryl hydrocarbon receptor AhR, thereby, displaying a plethora of biological effects. Biological activities of metoxylated and hydroxylated stilbenes were studied in the past. The aim of the current study was to describe the effects of 13 different hydroxy- and methoxystilbenes, including their cis/trans isomers on the transcriptional activity of AhR and the expression of CYP1A genes in hepatic cancer cells HepG2 and in primary human hepatocytes. Techniques of gene reporter assays, qRT-PCR, Simple Western blotting by Sally Sue™ and electrophoretic mobility shift assay EMSA were employed. All compounds activated AhR, but their efficacies, potencies and dose-response profiles differed substantially. The strongest activators of AhR and inducers of CYP1A1 in HepG2 cells were DMU-212 ((E)-3,4,5,4´-tetramethoxystilbene), trans-piceatannol, cis-piceatannol, trans-trismethoxyresveratrol and trans-pinostilbene. While DMU-212 and trans-trismethoxyresveratrol also induced CYP1A1 and CYP1A2 in primary human hepatocytes, the effects of trans-piceatannol, cis-piceatannol and trans-pinostilbene weaned off. On the other hand, trans-4-methoxystilbene was strong CYP1A inducer in hepatocytes but not in HepG2 cells. Differences between effects of stilbenes in HepG2 cells and human hepatocytes are probably due to the extensive phase I and phase II xenobiotic metabolism in human hepatocytes. The data obtained may be of toxicological relevance.
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Paraoxonase 1 (PON1) is a calcium-dependent lactonase synthesized primarily in the liver and secreted into the plasma, where it is associates with high density lipoproteins (HDL). PON1 acts as antioxidant preventing low-density lipoprotein (LDL) oxidation, a process considered critical in the initiation and progression of atherosclerosis. Additionally, PON1 hydrolyzes and detoxifies some toxic metabolites of organophosphorus compounds (OPs). Thus, PON1 activity and expression levels are important for determining susceptibility to OPs intoxication and risk of developing diseases related to inflammation and oxidative stress. Increasing evidence has demonstrated the modulation of PON1 expression by many factors is due to interaction with nuclear receptors (NRs). Here, we briefly review the studies in this area and discuss the role of nuclear receptors in the regulation of PON1 expression, as well as how understanding these mechanisms may allow us to manipulate PON1 levels to improve drug efficacy and treat disease.
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Nevirapine is associated with severe hepatotoxicity, through the formation of reactive metabolites. Paraoxonase-1 (PON-1) is a promiscuous enzyme involved in the metabolism of xeno- and endobiotics and proposed as a biomarker of hepatotoxicity. The aim of this work was to explore the effects of nevirapine and its phase I metabolites, 2-hydroxy-nevirapine and 12-hydroxy-nevirapine, on PON-1 activities.
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Effects of nevirapine and its metabolites were only observed in the 3D cell model. Both nevirapine and 12-hydroxy-nevirapine increased POase (p < 0.05, p < 0.01) and LACase activities (p < 0.05, p < 0.001). The AREase activity was increased only upon 12-hydroxy-nevirapine exposure (p < 0.01). These modulatory effects were observed at 300 μM concentrations of nevirapine and 12-hydroxy-nevirapine.
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¹: Both authors contribute equally to this work.

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Identification of a new stilbene-derived inducer of paraoxonase 1 and ligand of the Aryl hydrocarbon Receptor

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals

Cell culture

Results

Discussion

FEBS Lett

Pharmacol Rep

FEBS Lett

J Lipid Res

Biochem Pharmacol

Biochimie

Metabolism

Metabolism

Cell

Atherosclerosis

Atherosclerosis

The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin

Nat Genet

Decreased atherosclerotic lesion formation in human serum paraoxonase transgenic mice

Circulation

Paraoxonase 1 protects against protein N-homocysteinylation in humans

FASEB J

Human serum paraoxonase 1 decreases macrophage cholesterol biosynthesis: possible role for its phospholipase-A2-like activity and lysophosphatidylcholine formation

Arterioscler Thromb Vasc Biol