Paraoxonase 2 Research Articles

0501: Association of Cys311Ser polymorphism of paraoxonase-2 gene with myocardial infarction in the Tunisian male population

Paraoxonase 2 (PON2) has antioxidant properties similar to paraoxonase-1 and paraoxonase-3. However, in contrast to paraoxonase-1 and paraoxonase-3, paraoxonase-2 is not associated with high-density lipoproteins and may only exert its antioxidant function at the cellular level. PON2 may also play a role in the pathogenesis of arterial thrombosis and atherosclerosis. The purpose of the present study was to investigate the possible association between the Cys311Ser polymorphism of PON2 gene and myocardial infarction (MI) in the Tunisian male population A total of 700 unrelated Tunisian subjects including 321 patients with MI and 379 healthy controls were included in this study. Genomic DNA was extracted from peripheral blood leukocytes according to standard methods. PON2 genotypes were determined by PCRRFLP method. A significant difference in genotype distribution and allele frequencies was observed between MI patients and controls. Patients with MI had a frequency of 17.1% for CC genotype, 45.8% for the CS genotype and 37.1% for the SS genotype. The controls had a frequency of 26.1% for the CC genotype, 45.6% for the CS genotype and 28.2% for the SS genotype (χ 2 =10.58; p=0.005). The MI patient group showed a significantly higher frequency of the S allele compared to the controls (0.60 vs 0.51; χ 2 =11.16; p<0.001). In multivariate analysis, C311S polymorphism was independently associated with MI (p<0.001). Our results showed a significant and independent association between the PON2 C311S polymorphism (presence of S allele) and MI in the Tunisian male population.

DJ-1 interacts with and regulates paraoxonase-2, an enzyme critical for neuronal survival in response to oxidative stress.

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.

English

OBJECTIVE: Pre-eclampsia and atherosclerosis are both endothelial diseases with an involvement of lipid-mediated oxidative damage. The intracellular antioxidant enzyme paraoxonase 2 (PON2) may have a protective function in the prevention of atherogenesis. Oxidative stress have important role in the pathogenesis of pre-eclampsia. Study was designed to investigate monocytic paraoxonase 2 activity in pre-eclampsia. DESIGN AND METHOD: Study group was case-control study of 60 with preeclampsia and 60 uncomplicated term-deliveries. Paraoxonase2 activity done using substrate Dihydrocoumarin (DHC). RESULTS: Monocytic Paraoxonase2 activity was significantly lower in women with preeclampsia v/s controls (1.22U/mg versus1.67U/mg protein, p =0.002) Serum levels of T. chol, LDLc and VLDLc are higher in cases than in controls and are statistically significant. Serum nitric oxide also showed significant decrease in cases 22.92umol/L versus 25.12umol/L, p=0.015. In multivariate regression analysis PON2 (OR 2.393, p=0.002) and for nitric oxide (OR 1.091, p=0.030). Multivariate logistic regression analysis after adjustment of other risk factors demonstrates decreased PON2 lactonase activity is associated with greatest risk for preeclampsia. Model 1:, total cholesterol, HDL-C, LDL-C, nitric oxide ( R2=0.137, p= 0.011) , model 2 All parameters in Model I + PON2 lactonase activity. (R2=0.243, p<0.001). ROC plots demonstrated a high diagnostic accuracy for model 2 (area under ROC curve = 0.745) than model1 (area under ROC curve=0.686) CONCLUSION: decreased PON2 lactonase activity, abnormal lipid profile and decreased nitric oxide (nitrate+ nitrite) may have role in pathogenesis of pre-eclampsia and also gives valuable information regarding the risk prediction of pre-eclampsia.

Modulating reconstituted high density lipoprotein functionality to target the Pseudomonas aeruginosa quorum sensing system

AimsThe synthetic counterparts of serum high density lipoproteins (HDL; reconstituted HDL, reHDL) are assuming increasing importance as a therapeutic vector. They circulate not only in blood, but also outside the vascular compartment giving access to all body tissues. Presently, the therapeutic use of reHDL exploits inherent HDL functions. Our aim was to determine if HDL functionality could be modulated by attaching peptides not normally associated with the complex. Main methodsA peptide chimera was designed by linking the signal peptide of the HDL-associated enzyme paraoxonase-1 (PON1) to the coding region for the intracellular enzyme paraoxonase-2 (PON2). Key findingsThe signal peptide modified the properties of PON2, promoting its secretion from cells and binding to HDL. Enzyme activity of the chimera protein was highly stable. Conditioned HDL showed the functions of PON2 in its ability to hydrolyse typical PON2 substrates, namely homoserine lactones. Further in vitro studies showed that conditioned HDL was able to reduce the virulence of Pseudomonas aeruginosa. Both biofilm formation and the activation of the quorum sensing systems las and rhl, responsible for bacterial virulence, were significantly reduced. SignificanceThe study provides proof of principal that the signal peptide of PON1 can be used to attach peptides to HDL and thus modulate HDL function. They may provide a vector that is ubiquitously distributed in extracellular body fluids for designing therapeutic strategies to address different pathophysiological states.

Paraoxonase enzyme protects retinal pigment epithelium from chlorpyrifos insult.

Retinal pigment epithelium (RPE) provides nourishment and protection to the eye. RPE dysfunction due to oxidative stress and inflammation is one of the major reason for many of the retinal disorders. Organophosphorus pesticides are widely used in the agricultural, industrial and household activities in India. However, their effects on the eye in the context of RPE has not been studied. In this study the defense of the ARPE19 cells exposed to Chlorpyrifos (1 nM to 100 µM) in terms of the enzyme paraoxonase (PON) was studied at 24 hr and 9 days of treatment. Chlorpyrifos was found to induce oxidative stress in the ARPE19 cells as seen by significant increase in ROS and decrease in glutathione (GSH) levels without causing cell death. Tissue resident Paraoxonase 2 (PON2) mRNA expression was elevated with chlorpyrifos exposure. The three enzymatic activities of PON namely, paraoxonase (PONase), arylesterase (PON AREase) and thiolactonase (PON HCTLase) were also found to be significantly altered to detoxify and as an antioxidant defense. Among the transcription factors regulating PON2 expression, SP1 was significantly increased with chlorpyrifos exposure. PON2 expression was found to be crucial as ARPE19 cells showed a significant loss in their ability to withstand oxidative stress when the cells were subjected to chlorpyrifos after silencing PON2 expression. Treatment with N-acetyl cysteine positively regulated the PON 2 expression, thus promoting the antioxidant defense put up by the cells in response to chlorpyrifos.

Dopamine D1 and D5 receptors differentially regulate paraoxonase 2 (1134.6)

We have previously reported that dopamine D1‐like receptors (D1R and D5R) negatively regulate NADPH oxidase isoform expression and activity in renal proximal tubule (RPT) cells from rodents and humans. We tested the hypothesis that D1‐like dopamine receptors (D1R and D5R) differentially regulate the anti‐oxidant enzyme paraoxonase2 (PON2) in HEK‐293 cells heterologously expressing human D1R and D5R. Fenoldopam (1µM, 15 min), an agonist for both D1R and D5R, increased PON2 co‐immunoprecipitation with D1R (160±4% vs. control, 101±1%) and D5R (132±0.1% vs. control, 102±1%) in both cell lines. Short‐term (15, 30, and 60min) stimulation of D1R and D5R with fenoldopam (1µM) did not alter total PON2 protein. However, in D1R cells, at 15min, fenoldopam increased PON2 protein in both lipid rafts (LRs) (144±3% vs. control, 100±3%) and non‐LRs (163±25% vs. control, 100±21%) (P&lt;0.05, n=6/group, ANOVA). In contrast, in D5R cells, fenoldopam increased PON2 protein only in non‐LRs (132±5% vs. control, 100±1.6%, n=3). Long‐term (2, 4, 8, and 24h) fenoldopam (1µM) stimulation increased PON2 protein in a time dependent manner (146% ±7/8h, and 162% ±10/24h vs. control, 100±9%) (P&lt;0.05, n=4, ANOVA) in D5R but not D1R cells. Fenoldopam also increased PON2 mRNA (1.25‐fold) (n=3, P&lt;0.05, t‐test) at 24h in D5R but not D1R cells. Silencing PON2 gene with PON2‐siRNA (10nM/48h) in D5R cells decreased PON2 protein (‐40±4% vs. mock‐siRNA, 100±17%, n=3) and mRNA (‐17±4% vs. mock‐siRNA, 100±2%, P&lt;0.05, n=3, t‐test) and increased basal ROS production by 1.36‐fold, relative to mock‐siRNA. PON2 protein was decreased in D5R‐/‐ mice (‐44±13%) relative to D5R+/+ mice (100±8%, n=4‐6/group). We conclude that short‐term stimulation of either D1R or D5R leads to PON2 protein recruitment to non‐LRs and also to LRs in the case of D1R. However, long‐term stimulation of PON2 and negative ROS regulation are mainly due to D5R.Grant Funding Source: HL023081, HL074940, DK039308

Metformin inhibits macrophage cholesterol biosynthesis rate: Possible role for metformin-induced oxidative stress

The aim of the present study was to analyze the metformin (MF) effect on two cellular atherogenic activities: cholesterol biosynthesis and oxidative-stress (OS) as studied in J774A.1 macrophage cell line. MF (2–5 mM) significantly and dose – dependently reduced macrophage cholesterol content and cholesterol biosynthesis rate from acetate, but not from mevalonate, by up to 68% and 71%, respectively. MF inhibitory effect on cholesterol biosynthesis was similar to that of simvastatin. In contrast to the above anti-atherogenic MF effect, MF significantly increased cellular OS as shown by enhancement of reactive oxygen species (ROS) production by up to 70%, and decrement in cellular reduced glutathione (GSH) levels by up to 67%. Macrophage paraoxonase2 (PON2) expression however, increased by MF, by up to 1.5 folds. To overcome the MF oxidation stimulation, macrophages were incubated with MF together with potent dietary antioxidants, i.e. −5 μg GAE/ml of pomegranate juice (PJ) or 30 μM of vitamin E (VE). Both of these potent antioxidants substantially reduced MF-induced OS, and in parallel, abolished MF inhibitory effect on cholesterol biosynthesis rate. We thus conclude that the inhibition of macrophage cholesterol biosynthesis by MF is related, at least in part, to MF-induced OS.

Paraoxonase 2 Gene (Cys&amp;lt;sup&amp;gt;311&amp;lt;/sup&amp;gt;-Ser) Polymorphism and the Risk of Coronary Artery Disease

Background: Human paraoxonase-2(PON2) which is exclusively intracellular possesses unique properities that distinguish it from PON1 and PON3. Recently, it was demonstrated that PON2 protects against atherosclerosis by preventing LDL oxidation. Emerging evidences have proposed that genetic variations in the PON2 gene may be associated with coronary artery disease (CAD). Objectives: To investigate the relationship between a common PON2 gene (Cys311-Ser) polymorphism and the presence and extent of CAD. Methods: The study comprised 112 patients recruited from those undergoing coronary angiography for suspected CAD, who were divided according to the presence or absence of CAD into 2 groups Group I including 62 patients with CAD and Group II including 50 patients proved to have normal coronaries. All the subjects included in the study were genotyped for the (Cys311-Ser) polymorphism of PON2 gene using RCR-RFLP. Results: The frequency of Cys allele was significantly higher in group I compared to Group II (77.4% vs. 56% respectively, P 311 Cys allele [OR 5.67, CI (1.99 - 14.77), P

Association between single nucleotide polymorphisms of PON2 gene and susceptibility to occupational noise-induced deafness among Chinese Han population exposed to high noise levels

To investigate the association between the single nucleotide polymorphisms (SNPs) of paraoxonase-2 (PON2) gene and the susceptibility to occupational noise-induced deafness among Chinese Han population exposed to high noise levels [>85 dB (A)]. A case-control study was conducted in Chinese Han population exposed to high noise levels. The subjects were divided into case group (n = 127) and control group (n = 136) according to the Diagnostic criteria of occupational noise-induced deafness (GBZ 49-2007). The case group was composed of 127 workers with a mean binaural high-frequency hearing threshold not less than 40 dB, as measured using an electro-audiometer, while the control group was composed of 136 workers with a mean binaural high-frequency hearing threshold less than 40 dB, as measured using the electro-audiometer, who were on shift in the same position as the cases and matched with them for age, sex, and years of noise exposure. Peripheral venous blood (2 ml) was collected from each subject during physical examination to extract genomic DNA, and genotypes were identified using a TaqMan probe. PON2 genotypes rs7493 CG+GG, rs7785846 CT+TT, rs12026 CG+GG, and rs7786401 GT+TT were the risk factors for occupational noise-induced deafness, and the adjusted odds ratios (95%confidence intervals) were 5.87 (3.11∼11.07), 5.92 (3.10∼11.32), 5.53 (2.93∼10.45), and 5.93 (3.10∼11.34), respectively. In addition, the higher the noise exposure levels, the higher the risk of developing occupational noise-induced deafness among the individuals carrying mutant genotypes. PON2 genotypes rs7493 CG+GG, rs7785846 CT+TT, rs12026 CG+GG, and rs7786401 GT +TT may be associated with the susceptibility to occupational noise-induced deafness among Chinese Han population exposed to high noise levels, and the effects of mutant genotypes and noise exposure levels may be mutually enhanced.

Paraoxonase-2 (PON2) in brain and its potential role in neuroprotection

Paraoxonase 2 (PON2) is a member of a gene family which also includes the more studied PON1, as well as PON3. PON2 is unique among the three PONs, as it is expressed in brain tissue. PON2 is a lactonase and displays anti-oxidant and anti-inflammatory properties. PON2 levels are highest in dopaminergic regions (e.g. striatum), are higher in astrocytes than in neurons, and are higher in brain and peripheral tissues of female mice than male mice. At the sub-cellular level, PON2 localizes primarily in mitochondria, where it scavenges superoxides. Lack of PON2 (as in PON2−/− mice), or lower levels of PON2 (as in male mice compared to females) increases susceptibility to oxidative stress-induced toxicity. Estradiol increases PON2 expression in vitro and in vivo, and provides neuroprotection against oxidative stress. Such neuroprotection is not present in CNS cells from PON2−/− mice. Similar results are also found with the polyphenol quercetin. PON2, given its cellular localization and antioxidant and anti-inflammatory actions, may represent a relevant enzyme involved in neuroprotection, and may represent a novel target for neuroprotective strategies. Its differential expression in males and females may explain gender differences in the incidence of various diseases, including neurodevelopmental, neurological, and neurodegenerative diseases.

Metformin inhibits macrophage cholesterol biosynthesis rate: Possible role for metformin-induced oxidative stress

The aim of the present study was to analyze the metformin (MF) effect on two cellular atherogenic activities: cholesterol biosynthesis and oxidative-stress (OS) as studied in J774A.1 macrophage cell line. MF (2–5mM) significantly and dose-dependently reduced macrophage cholesterol content and cholesterol biosynthesis rate from acetate, but not from mevalonate, by up to 68% and 71%, respectively. MF inhibitory effect on cholesterol biosynthesis was similar to that of simvastatin. In contrast to the above anti-atherogenic MF effect, MF significantly increased cellular OS as shown by enhancement of reactive oxygen species (ROS) production by up to 70%, and decrement in cellular reduced glutathione (GSH) levels by up to 67%. Macrophage paraoxonase2 (PON2) expression however, increased by MF, by up to 1.5 folds. To overcome the MF oxidation stimulation, macrophages were incubated with MF together with potent dietary antioxidants, i.e. −5μg GAE/ml of pomegranate juice (PJ) or 30μM of vitamin E (VE). Both of these potent antioxidants substantially reduced MF-induced OS, and in parallel, abolished MF inhibitory effect on cholesterol biosynthesis rate. We thus conclude that the inhibition of macrophage cholesterol biosynthesis by MF is related, at least in part, to MF-induced OS.

Renal Dopamine Receptors, Oxidative Stress, and Hypertension

Dopamine, which is synthesized in the kidney, independent of renal nerves, plays an important role in the regulation of fluid and electrolyte balance and systemic blood pressure. Lack of any of the five dopamine receptor subtypes (D1R, D2R, D3R, D4R, and D5R) results in hypertension. D1R, D2R, and D5R have been reported to be important in the maintenance of a normal redox balance. In the kidney, the antioxidant effects of these receptors are caused by direct and indirect inhibition of pro-oxidant enzymes, specifically, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase, and stimulation of anti-oxidant enzymes, which can also indirectly inhibit NADPH oxidase activity. Thus, stimulation of the D2R increases the expression of endogenous anti-oxidants, such as Parkinson protein 7 (PARK7 or DJ-1), paraoxonase 2 (PON2), and heme oxygenase 2 (HO-2), all of which can inhibit NADPH oxidase activity. The D5R decreases NADPH oxidase activity, via the inhibition of phospholipase D2, and increases the expression of HO-1, another antioxidant. D1R inhibits NADPH oxidase activity via protein kinase A and protein kinase C cross-talk. In this review, we provide an overview of the protective roles of a specific dopamine receptor subtype on renal oxidative stress, the different mechanisms involved in this effect, and the role of oxidative stress and impairment of dopamine receptor function in the hypertension that arises from the genetic ablation of a specific dopamine receptor gene in mice.

Role of PON2 in innate immune response in an acute infection model

N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) is a quorum-sensing molecule produced by gram-negative microbial pathogens such as Pseudomonas aeruginosa (PAO1). 3OC12-HSL is involved in the regulation of bacterial virulence factors and also alters the function of the host immune cells. Others and we have previously shown that paraoxonase 2 (PON2), a member of the paraoxonase gene family expressed in immune cells, hydrolyzes 3OC12-HSL. In this study, we examined i) whether macrophage PON2 participates in 3OC12-HSL hydrolysis, ii) the effect of PON2 deficiency in acute PAO1 infection in mice and iii) the effect of 3OC12-HSL on PON2 deficient (PON2-def) macrophages. When compared to wild type macrophages, both intact cells and membrane-enriched protein lysates obtained from PON2-def macrophages show a marked impairment in their ability to hydrolyze 3OC12-HSL. PON2 expression (message and protein) is not altered in response to 3OC12-HSL in macrophages. 3OC12-HSL treated PON2-def macrophages showed i) an increase in ER stress and oxidative stress, ii) defective phosphatidylinositol 3-kinase (PI3 kinase)/AKT activation, and iii) reduced phagocytosis function. Moreover, the nitration to phosphorylation ratio of Tyr458 in p85 protein, the regulatory subunit of PI3-kinase that has been correlated with the phagocytosis function of macrophages, was increased in PON2-def macrophages. Antioxidant treatment reversed the effects of PON2 deficiency in macrophage phagocytosis function. Furthermore, following administration of 1.6×107 CFU of PAO1, bacterial clearance was significantly reduced in the lungs (5.7 fold), liver (2.5 fold), and spleen (14.8 fold) of PON2-def mice when compared to wild type mice. Our results suggest that PON2 plays an important role in innate immune defense against PAO1 infection.

Modulation of Paraoxonase 2 (PON2) in Mouse Brain by the Polyphenol Quercetin: A Mechanism of Neuroprotection?

Quercetin is a common flavonoid polyphenol which has been shown to exert neuroprotective actions in vitro and in vivo. Though quercetin has antioxidant properties, it has been suggested that neuroprotection may be ascribed to its ability of inducing the cell's own defense mechanisms. The present study investigated whether quercetin could increase the levels of paraoxonase 2 (PON2), a mitochondrial enzyme expressed in brain cells, which has been shown to have potent antioxidant properties. PON2 protein, mRNA, and lactonase activity were highest in mouse striatal astrocytes. Quercetin increased PON2 levels, possibly by activating the JNK/AP-1 pathway. The increased PON2 levels induced by quercetin resulted in decreased oxidative stress and ensuing toxicity induced by two oxidants. The neuroprotective effect of quercetin was significantly diminished in cells from PON2 knockout mice. These findings suggest that induction of PON2 by quercetin represents an important mechanism by which this polyphenol may exert its neuroprotective action.

Abstract 560: Role of Paraoxonase 2 In Innate Immune Response in an Acute Infection Model

Background (3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a quorum-sensing molecule produced by certain classes of gram-negative microbial pathogens, such as Pseudomonas aeruginosa (PAO1), is involved in the regulation of virulence factors and alters the function of the host cells. Previously, We have shown that paraoxonase 2 (PON2) hydrolyzes 3OC12-HSL in murine tracheal epithelial cells. In this study, we examined the physiological role of PON2 in innate host defense using the PA01-induced acute infection mouse model. Materials and Results We demonstrates that both intact cells and membrane-enriched protein lysates obtained from PON2-deficient (PON2-def) macrophages reveal a marked impairment in their ability to hydrolyze 3OC12-HSL. PON2 is neither up-regulated nor down-regulated in response to 3OC12-HSL in control macrophages. In an ex vivo model, macrophages derived from PON2-def mice show an increase in ER stress-mediated oxidative stress and defective PI3 kinase/AKT activation, leading to reduced phagocytes function compared to control macrophages following 3OC12-HSL treatment. Moreover, p85, a regulatory subunit of PI3-kinase, shows higher tyrosine nitration in the macrophage of PON2-def mice which is associated with phagocytic function. Restoration of PON2 in the macrophages reduces the ER stress mediated oxidative stress and improves the phagocytosis function in response to 3OC12-HSL treatment. Furthermore, following administration of 1.6x10 7 CFU of PAO1, a decrease in bacterial clearance is found in the lungs, and liver of PON2-def mice by 5.7, and 14.8 fold, respectively. Conclusion Increased ER stress mediated oxidative stress decreases phagocytosis function in the macrophages of PON2-def mice in response to 3OC12-HSL may explain the decreased bacterial clearance observed in PON2-def mice following PAO1 infection.

Monocyte-macrophage membrane possesses free radicals scavenging activity: stimulation by polyphenols or by paraoxonase 1 (PON1)

In the current study, we analysed free radicals scavenging activity of monocytes-macrophages in the absence or presence of antioxidants such as polyphenols or paraoxonase 1 (PON1). THP-1 human monocytic cell line, murine J774A.1 macrophages, as well as human primary monocytes have the capability to scavenge free radicals, as measured by the 1-diphenyl-2-picryl-hydrazyl (DPPH) assay. This effect (which could be attributed to the cell's membrane) was cell number and incubation time dependent. Upon incubation of J774A.1 macrophages with acetylated LDL (Ac-LDL), with VLDL, or with the radical generator, AAPH, the cells’ lipid peroxides content, and paraoxonase 2 (PON2) activity were significantly increased. While non-treated cells decreased DPPH absorbance by 65%, the Ac-LDL-, VLDL- or AAPH-treated cells, decreased it by only 33%, 30%, or 45%, respectively. We next analysed the effect of J774A.1 macrophage enrichment with antioxidants, such as polyphenols or PON1 on the cells’ free radicals scavenging activity. Non-treated cells decreased DPPH absorbance by 50%, whereas vitamin E-, punicalagin- or PJ-treated cells significantly further decreased it, by 75%. Similarly, in PON1-treated cells DPPH absorbance was further decreased by 63%, in association with 23% increment in PON1 catalytic activity. In cells under oxidative stress [treated with AAPH-, or with oxidized LDL], PON1 activity was decreased by 31% or 40%, as compared to the activity observed in PON1 incubated with non-treated cells. We conclude that monocytes-macrophages possess free radicals scavenging activity, which is decreased under atherogenic conditions, and increased upon cell enrichment with potent antioxidants such as nutritional polyphenols, or PON1.

Gender differences in brain susceptibility to oxidative stress are mediated by levels of paraoxonase-2 expression

Paraoxonase 2 (PON2), a member of a gene family that also includes PON1 and PON3, is expressed in most tissues, including the brain. In mouse brain, PON2 levels are highest in dopaminergic areas (e.g., striatum) and are higher in astrocytes than in neurons. PON2 is primarily located in mitochondria and exerts a potent antioxidant effect, protecting mouse CNS cells against oxidative stress. The aim of this study was to characterize PON2 expression and functions in the brains of male and female mice. Levels of PON2 (protein, mRNA, and lactonase activity) were higher in brain regions and cells of female mice. Astrocytes and neurons from male mice were significantly more sensitive (by 3- to 4-fold) to oxidative stress-induced toxicity than the same cells from female mice. Glutathione levels did not differ between genders. Importantly, no significant gender differences in susceptibility to the same oxidants were seen in cells from PON2−/− mice. Treatment with estradiol induced a time- and concentration-dependent increase in the levels of PON2 protein and mRNA in male (4.5-fold) and female (1.8-fold) astrocytes, which was dependent on activation of estrogen receptor-α. In ovariectomized mice, PON2 protein and mRNA were decreased to male levels in brain regions and in liver. Estradiol protected astrocytes from wild-type mice against oxidative stress-induced neurotoxicity, but did not protect cells from PON2−/− mice. These results suggest that PON2 is a novel major intracellular factor that protects CNS cells against oxidative stress and confers gender-dependent susceptibility to such stress. The lower expression of PON2 in males may have broad ramifications for susceptibility to diseases involving oxidative stress, including neurodegenerative diseases.

Triglyceride accumulation in macrophages upregulates paraoxonase 2 (PON2) expression via ROS‐mediated JNK/c‐Jun signaling pathway activation

The aim of this study was to analyze the effect and mechanism of action of macrophage triglyceride accumulation on cellular PON2 expression. Incubation of J774A.1 (murine macrophages) with VLDL (0-75 μg protein/mL) significantly and dose-dependently increased cellular triglyceride mass, and reactive oxygen species (ROS) formation, by up to 3.3- or 1.8-fold, respectively. PON2 expression (mRNA, protein, activity) in cells treated with VLDL (50 μg protein/mL) was higher by 2- to 3-fold, as compared with control cells. Similar effects were noted upon using THP-1 (human macrophages). Incubation of macrophages with synthetic triglyceride or triglyceride fraction from carotid lesion resulted in similar effects, as shown for VLDL. Upon using specific inhibitors of MEK1/2 (UO126, 10 μM), p38 (SB203580, 10 μM), or JNK (SP600125, 20 μM), we demonstrated that MEK, as well as JNK, but not p38, are involved in VLDL-induced macrophage PON2 upregulation. VLDL activated JNK (but not ERK), which resulted in c-Jun phosphorylation. This signaling pathway is probably activated by ROS, since the antioxidant reduced glutathione (GSH), significantly decreased VLDL-induced macrophage ROS formation, c-Jun phosphorylation and PON2 overexpression. We conclude that macrophage triglyceride accumulation upregulates PON2 expression via MEK/ JNK/c-Jun pathway, and these effects could be related, at least in part, to cellular triglycerides-induced ROS formation. ©

Paraoxonase 2 decreases renal reactive oxygen species production, lowers blood pressure, and mediates dopamine D2 receptor-induced inhibition of NADPH oxidase

The dopamine D2 receptor (D2R) regulates renal reactive oxygen species (ROS) production, and impaired D2R function results in ROS-dependent hypertension. Paraoxonase 2 (PON2), which belongs to the paraoxonase gene family, is expressed in various tissues, acting to protect against cellular oxidative stress. We hypothesized that PON2 may be involved in preventing excessive renal ROS production and thus may contribute to maintenance of normal blood pressure. Moreover, D2R may decrease ROS production, in part, through regulation of PON2. D2R colocalized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (−33±6%) in D2−/− relative to D2+/+ mice. Renal subcapsular infusion of PON2 siRNA decreased PON2 protein expression (−55%), increased renal oxidative stress (2.2-fold), associated with increased renal NADPH oxidase expression (Nox1, 1.9-fold; Nox2, 2.9-fold; and Nox4, 1.6-fold) and activity (1.9-fold), and elevated arterial blood pressure (systolic, 134±5 vs 93±6mmHg; diastolic, 97±4 vs 65±7mmHg; mean 113±4 vs 75±7mmHg). To determine the relevance of the PON2 and D2R interaction in humans, we studied human renal proximal tubule cells. Both D2R and PON2 were found in nonlipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D2R/D3R agonist quinpirole (1μM, 24h) decreased ROS production (−35±6%), associated with decreased NADPH oxidase activity (−32±3%) and expression of Nox2 (−41±7%) and Nox4 (−47±8%) protein, and increased expression of PON2 mRNA (2.1-fold) and protein (1.6-fold) at 24h. Silencing PON2 (siRNA, 10nM, 48h) not only partially prevented the quinpirole-induced decrease in ROS production by 36%, but also increased basal ROS production (1.3-fold), which was associated with an increase in NADPH oxidase activity (1.4-fold) and expression of Nox2 (2.1-fold) and Nox4 (1.8-fold) protein. Inhibition of NADPH oxidase with diphenylene iodonium (10μM/30 min) inhibited the increase in ROS production caused by PON2 silencing. Our results suggest that renal PON2 is involved in the inhibition of renal NADPH oxidase activity and ROS production and contributes to the maintenance of normal blood pressure. PON2 is positively regulated by D2R and may, in part, mediate the inhibitory effect of renal D2R on NADPH oxidase activity and ROS production.

A Diet Rich in Olive Oil Phenolics Reduces Oxidative Stress in the Heart of SAMP8 Mice by Induction of Nrf2-Dependent Gene Expression

A Mediterranean diet rich in olive oil has been associated with health benefits in humans. It is unclear if and to what extent olive oil phenolics may mediate these health benefits. In this study, we fed senescence-accelerated mouse-prone 8 (SAMP8, n=11 per group) semisynthetic diets with 10% olive oil containing either high (HP) or low amounts of olive oil phenolics (LP) for 4.5 months. Mice consuming the HP diet had significantly lower concentrations of the oxidative damage markers thiobarbituric acid-reactive substances and protein carbonyls in the heart, whereas proteasomal activity was similar in both groups. Nrf2-dependent gene expression may be impaired during the aging process. Therefore, we measured Nrf2 and its target genes glutathione-S-transferase (GST), γ-glutamyl cysteine synthetase (γ-GCS), nicotinamide adenine dinucleotide phosphate [NAD(P)H]:quinone oxidoreductase (NQO1), and paraoxonase-2 (PON2) in the hearts of these mice. Nrf2 as well as GST, γ-GCS, NQO1, and PON2 mRNA levels were significantly higher in heart tissue of the HP as compared to the LP group. The HP-fed mice had significantly higher PON1 activity in serum compared to those receiving the LP diet. Furthermore, HP feeding increased relative SIRT1 mRNA levels. Additional mechanistic cell culture experiments were performed, and they suggest that the olive oil phenolic hydroxytyrosol present in the HP oil may be responsible for the induction of Nrf2-dependent gene expression and the increase in PON activity. In conclusion, a diet rich in olive oil phenolics may prevent oxidative stress in the heart of SAMP8 mice by modulating Nrf2-dependent gene expression.