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1.  Impaired hepatic insulin signalling in PON2-deficient mice: a novel role for the PON2/apoE axis on the macrophage inflammatory response 
The Biochemical journal  2011;436(1):91-100.
Hepatic glucose metabolism is strongly influenced by oxidative stress and pro-inflammatory stimuli. PON2 (paraoxonase 2), an enzyme with undefined antioxidant properties, protects against atherosclerosis. PON2-deficient (PON2-def) mice have elevated hepatic oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages. In the present paper, we demonstrate that PON2 deficiency is associated with inhibitory insulin-mediated phosphorylation of hepatic IRS-1 (insulin receptor substrate-1). Unexpectedly, we observed a marked improvement in the hepatic IRS-1 phosphorylation state in PON2-def/apoE (apolipoprotein E)−/− mice, relative to apoE−/− mice. Factors secreted from activated macrophage cultures derived from PON2-def and PON2-def/apoE−/− mice are sufficient to modulate insulin signalling in cultured hepatocytes in a manner similar to that observed in vivo. We show that the protective effect on insulin signalling in PON2-def/apoE−/− mice is directly associated with altered production of macrophage proinflammatory mediators, but not elevated intracellular oxidative stress levels. We further present evidence that modulation of the macrophage inflammatory response in PON2-def/apoE−/− mice is mediated by a shift in the balance of NO and ONOO− (peroxynitrite) formation. Our results demonstrate that PON2 plays an important role in hepatic insulin signalling and underscores the influence of macrophage-mediated inflammatory response on hepatic insulin sensitivity.
PMCID: PMC3743554  PMID: 21361875
apolipoprotein E (apoE); insulin receptor substrate-1 (IRS-1); liver; macrophage; paraoxonase; paraoxonase 2 (PON2)
2.  Regulation of Hepatic Paraoxonase-1 Expression 
Journal of Lipids  2012;2012:684010.
Serum paraoxonase-1 (PON1) is a member of the paraoxonases family (PON1, PON2, and PON3). PON1 is synthesized and secreted by the liver, and in circulation it is associated with HDL. PON1 has antioxidative properties, which are associated with the enzyme's capability to decrease oxidative stress in atherosclerotic lesions and to attenuate atherosclerosis development. Epidemiological evidence demonstrates that low PON1 activity is associated with increased risk of cardiovascular events and cardiovascular disease and is an independent risk factor for coronary artery disease. Therefore, pharmacological modulation of PON1 activity or PON1 gene expression could constitute a useful approach for preventing atherosclerosis. A primary determinant of serum PON1 levels is the availability of the enzyme for release by the liver, the principal site of PON1 production. Together with the enzyme secretion rate, enzymatic turnover, and protein stability, the level of PON1 gene expression is a major determinant of PON1 status. This paper summarizes recent progress in understanding the regulation of PON1 expression in hepatocytes.
PMCID: PMC3324161  PMID: 22548179
3.  Macrophage Paraoxonase 2 regulates calcium homeostasis and cell survival under Endoplasmic Reticulum stress conditions and is sufficient to prevent the development of aggravated atherosclerosis in Paraoxonase 2 deficiency/apoE−/− mice on a Western diet 
Molecular genetics and metabolism  2012;107(3):416-427.
Paraoxonase 2 deficiency (PON2-def) alters mitochondrial function and exacerbates the development of atherosclerosis in mice. PON2 overexpression protects against ER stress in cell culture. In this paper, we examined the role of PON2 in the unexplored link between ER stress and mitochondrial dysfunction and tested whether restoration of PON2 in macrophages is sufficient to reduce aggravated atherosclerosis in PON2-def/apoE−/− mice on a Western diet. ER stress response genes, intracellular calcium levels, and apoptotic nuclei were significantly elevated in PON2-def/apoE−/− macrophages compared to apoE−/− macrophages in response to ER stressors, but not at the basal level. In contrast, PON2-def/apoE−/− macrophages exhibited greater mitochondrial stress at the basal level, which was further worsened in response to ER stressors. There was no difference in ER stress response genes and apoptotic nuclei between apoE−/− and PON2-def/apoE−/− macrophages when pretreated with xestospongin (which blocks the release of calcium from ER) suggesting that PON2 modulates cell survival and ER stress by maintaining calcium homeostasis. Treatment with a mitochondrial calcium uptake inhibitor, RU360, attenuated ER stressor mediated mitochondrial dysfunction in PON2-def/apoE−/− macrophages. CHOP expression (ER stress marker) and apoptotic nuclei were significantly higher in aortic lesions of PON2-def/apoE−/− mice compared to apoE−/− mice fed a Western diet. Restoration of PON2 in macrophage reduced ER stress, mitochondrial dysfunction and apoptosis in response to ER stressors. Furthermore, restoration of PON2 in macrophages reduced lesional apoptosis and atherosclerosis in PON2-def/apoE−/− mice on a Western diet. Our data suggest that macrophage PON2 modulates mechanisms that link ER stress, mitochondrial dysfunction and the development of atherosclerosis.
PMCID: PMC3483415  PMID: 22864055
Paraoxonase 2; Mitochondria; Endoplasmic reticulum; Macrophages; Oxidative stress
4.  Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex 
The Journal of Clinical Investigation  2013;123(9):3815-3828.
Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein–associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr71), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each other’s function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.
PMCID: PMC3754253  PMID: 23908111
5.  The Human Paraoxonase Gene Cluster As a Target in the Treatment of Atherosclerosis 
Antioxidants & Redox Signaling  2012;16(6):597-632.
The paraoxonase (PON) gene cluster contains three adjacent gene members, PON1, PON2, and PON3. Originating from the same fungus lactonase precursor, all of the three PON genes share high sequence identity and a similar β propeller protein structure. PON1 and PON3 are primarily expressed in the liver and secreted into the serum upon expression, whereas PON2 is ubiquitously expressed and remains inside the cell. Each PON member has high catalytic activity toward corresponding artificial organophosphate, and all exhibit activities to lactones. Therefore, all three members of the family are regarded as lactonases. Under physiological conditions, they act to degrade metabolites of polyunsaturated fatty acids and homocysteine (Hcy) thiolactone, among other compounds. By detoxifying both oxidized low-density lipoprotein and Hcy thiolactone, PONs protect against atherosclerosis and coronary artery diseases, as has been illustrated by many types of in vitro and in vivo experimental evidence. Clinical observations focusing on gene polymorphisms also indicate that PON1, PON2, and PON3 are protective against coronary artery disease. Many other conditions, such as diabetes, metabolic syndrome, and aging, have been shown to relate to PONs. The abundance and/or activity of PONs can be regulated by lipoproteins and their metabolites, biological macromolecules, pharmacological treatments, dietary factors, and lifestyle. In conclusion, both previous results and ongoing studies provide evidence, making the PON cluster a prospective target for the treatment of atherosclerosis. Antioxid. Redox Signal. 16, 597–632.
I. Introduction
II. The PON Family
A. Evolution of the PON genes
B. Structure of the PONs
1. Primary structure
2. Three-dimensional structure
C. Tissue and cellular distribution of PONs
D. Substrates of PONs
1. Chemical substrates
2. Physiological substrates
III. PONs Protect Against Atherosclerosis and Coronary Heart Disease by Decreasing the Toxicity of LDL
A. Atherosclerosis-related CVDs do great harm to human health
B. OxLDL is one of the most important risk factors for atherogenesis
C. PON1 contributes to the anti-atherosclerotic functions of HDL
D. PON activity decreases atherosclerosis
1. PON1 and its protection against atherosclerosis
2. PON2 and its protection against atherosclerosis
3. PON3 and atherosclerosis
4. Roles of the PON gene cluster in atherosclerosis
IV. PONs Protect Against Hcy Toxicity and Associated Atherogenesis
A. Hcy is an important risk factor in atherosclerosis
B. PONs protect against Hcy toxicity
V. PONs Polymorphisms and Coronary Artery Disease
A. PON1 polymorphisms
1. PON1 polymorphisms and PON1 activity
2. PON1 polymorphisms and plasma lipoprotein levels
3. PON1 polymorphisms and CHD
B. PON2 polymorphisms
1. PON2 polymorphisms
2. PON2 polymorphisms and plasma lipoprotein levels
3. PON2 polymorphisms and CHD
VI. Other Diseases Related to PONs
A. PONs' relations to diabetes mellitus and corresponding complications
B. PONs' associations with metabolic syndrome and obesity
C. PONs and aging
D. Other diseases related to PONs
VII. Regulation of PONs
A. Transcriptional regulation of PONs
B. Lipoproteins and their metabolic products regulate PONs expression
C. Biological macromolecules modulate PONs expression
D. Pharmacological modulators of PONs
1. Statins
2. Fibrates
3. Other cardiovascular drugs
4. Diabetic drugs
5. Other drugs
E. Dietary factors and lifestyle factors
1. Vitamin C and vitamin E
2. Natural plant extracts
3. Dietary polyphenol compounds
4. Dietary flavonoids
5. Dietary lipids
6. Alcohol
7. Cigarettes
8. Fasting
VIII. PONs Mimetics
IX. Conclusions and Future Directions
PMCID: PMC3270057  PMID: 21867409
6.  Temporal and Tissue-Specific Patterns of Pon3 Expression in Mouse: In situ Hybridization Analysis 
PON3 is a member of the paraoxonase gene family that includes PON1 and PON2. For example, PON3 and PON1 share approximately 60% identity at the amino acid level. Recent studies have demonstrated that PON3 is present in human and rabbit HDL but not in mouse HDL. Mouse PON3 appears to be cell-associated and is expressed in a wide range of tissues such as liver, adipose, macrophage, and the artery wall. In vitro studies have shown that PON3 can prevent LDL oxidation and destroy bacterial quorum-sensing molecules. Previous studies also showed that human PON3 transgenic mice were protected from obesity and atherosclerosis in both the C57BL/6J wild-type and LDLR knockout genetic background. Administration of adenovirus expressing the human PON3 gene into apoE −/− mice also decreased atherosclerotic lesion formation. In order to further understand the functions of PON3 in physiology and disease, we performed in situ hybridization analysis to examine Pon3 gene expression patterns in newborn and adult mice, in various tissues, including atherosclerotic lesions of apoE −/− mice. Our results show relatively high levels of Pon3 mRNA labeling in the adrenal gland, submaxillary gland, lung, liver, adipose, pancreas, large intestine, and other tissues of newborn mice. In the adult mouse, Pon3 mRNA levels were much lower in the corresponding tissues as mentioned above for the newborn mouse. Sections of the aortic root from the hearts of both wild-type and apoE −/− mice displayed moderate levels of Pon3 mRNA labeling. Pon3 mRNA was also detected in the atherosclerotic lesion areas at the aortic root of apoE −/− hearts. Our data revealed that mouse Pon3 is expressed in a wide range of tissues, and that its expression is temporally controlled.
PMCID: PMC3018859  PMID: 20221872
PON3; In situ hybridization; Developmental regulation; Mouse
7.  Reduced Paraoxonase 1 Activity as a Marker for Severe Coronary Artery Disease 
Disease markers  2013;35(2):97-103.
Paraoxonase-1 (PON1), a high-density-lipoprotein- (HDL-) associated enzyme, has the potential to protect against atherogenesis. We examine the relationships between plasma PON1 activity and the progression of atherosclerosis as well as coronary artery disease (CAD). Fasting blood samples were collected from female apolipoprotein E-deficient (apoE−/−) mice and 149 patients undergoing coronary angiography for the biochemical parameters measurement. The severity of CAD was defined using angiographic Gensini score (GSS). Compared to 3-month-old apoE−/− mice, aged mice had significantly lower PON1 activity, which is negatively correlated with the size of atherosclerotic lesion and plasma interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) levels. In study patients, PON1 activity was correlated with age, sex, and HDL-cholesterol, apolipoprotein AI, and high-sensitivity C-reactive protein (hs-CRP) levels and was significantly lower in CAD group than that in non-CAD control group. Interestingly, PON1 activity in severe CAD group (GSS > 40) was further significantly reduced compared to those in mild and moderate subgroups (GSS  ≤ 40) (P < 0.01). There is a significant correlation between PON1 activity and the severity of CAD as assessed by GSS (r = −0.393, P < 0.001). PON1 activity may be a potential biomarker for the severity of CAD.
PMCID: PMC3774974  PMID: 24167353
8.  In vivo administration of BL-3050: highly stable engineered PON1-HDL complexes 
Serum paraoxonase (PON1) is a high density lipoprotein (HDL)-associated enzyme involved in organophosphate (OP) degradation and prevention of atherosclerosis. PON1 comprises a potential candidate for in vivo therapeutics, as an anti-atherogenic agent, and for detoxification of pesticides and nerve agents. Because human PON1 exhibits limited stability, engineered, recombinant PON1 (rePON1) variants that were designed for higher reactivity, solubility, stability, and bacterial expression, are candidates for treatment. This work addresses the feasibility of in vivo administration of rePON1, and its HDL complex, as a potentially therapeutic agent dubbed BL-3050.
For stability studies we applied different challenges related to the in vivo disfunctionalization of HDL and PON1 and tested for inactivation of PON1's activity. We applied acute, repetitive administrations of BL-3050 in mice to assess its toxicity and adverse immune responses. The in vivo efficacy of recombinant PON1 and BL-3050 were tested with an animal model of chlorpyrifos-oxon poisoning.
Inactivation studies show significantly improved in vitro lifespan of the engineered rePON1 relative to human PON1. Significant sequence changes relative to human PON1 might hamper the in vivo applicability of BL-3050 due to adverse immune responses. However, we observed no toxic effects in mice subjected to repetitive administration of BL-3050, suggesting that BL-3050 could be safely used. To further evaluate the activity of BL-3050 in vivo, we applied an animal model that mimics human organophosphate poisoning. In these studies, a significant advantages of rePON1 and BL-3050 (>87.5% survival versus <37.5% in the control groups) was observed. Furthermore, BL-3050 and rePON1 were superior to the conventional treatment of atropine-2-PAM as a prophylactic treatment for OP poisoning.
In vitro and in vivo data described here demonstrate the potential advantages of rePON1 and BL-3050 for treatment of OP toxicity and chronic cardiovascular diseases like atherosclerosis. The in vivo data also suggest that rePON1 and BL-3050 are stable and safe, and could be used for acute, and possibly repeated treatments, with no adverse effects.
PMCID: PMC2785756  PMID: 19922610
9.  Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase. 
Journal of Clinical Investigation  1998;101(8):1581-1590.
HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.
PMCID: PMC508738  PMID: 9541487
10.  The Role of ‘Paraoxonase-1 Activity’ as an Antioxidant in Coronary Artery Diseases 
Background and Objectives: A majority of the Coronary Artery Diseases (CAD) result from complications of atherosclerosis. There is a growing body of evidence which has revealed that the reduced activity of the HDL-associated enzyme, paraoxonase1 (PON1), is predictive of vascular disease in humans, which include the results from prospective studies. The mechanisms by which PON1 activity influences risk of vascular disease continue to be evaluated. It is generally thought that PON1 contributes to the antioxidant, and thus, to the antiatherogenic properties of High Density Lipoproteins (HDL). Depleted antioxidant levels could be a risk factor for coronary artery disease. Hence, this study was done to evaluate PON1, as antioxidant, in CAD patients.
Methods: This study was done to determine serum levels of PON1 activity in 50 controls and in 60 clinically and ECG proven CAD cases and to compare PON1 activity with total cholesterol and triglycerides.
Results: Serum levels of PON1 activity (p<0.001) were significantly lower in CAD cases than in controls. Serum total cholesterol (p< 0.001) and triglyceride (p< 0.001) levels were significantly higher in CAD cases than in controls. There was a negative correlation between PON 1 activity and total cholesterol and triglycerides. The negative correlation between PON1 activity and total cholesterol was significant (p<0.05).
Interpretation and Conclusion: From our present study, we can conclude that PON1 can exert a protective effect on HDL by preventing its oxidative damage. Further, a decreased PON 1 activity may be a risk factor for CAD, which is likely to be explained by derangement of PON 1 activity towards lipid peroxidation. This study suggested that serum antioxidant activity of PON1 was an important factor which provided protection from oxidative stress and lipid peroxidation in CAD. Thus, evaluating the effects of PON 1 for CAD patients may be promising in the treatment and prognosis of CAD.
PMCID: PMC3749616  PMID: 23998046
Antioxidants; Coronary artery disease; Oxidative stress; Paraoxonase1
11.  Functionally Defective High-Density Lipoprotein and Paraoxonase: A Couple for Endothelial Dysfunction in Atherosclerosis 
Cholesterol  2013;2013:792090.
The endothelium is the primary target for biochemical or mechanical injuries caused by the putative risk factors of atherosclerosis. Endothelial dysfunction represents the ultimate link between atherosclerotic risk factors that promote atherosclerosis. HDL-C is thought to exert at least some parts of its antiatherogenic facilities via stimulating endothelial NO production, nearby inhibiting oxidative stress and inflammation. HDL-C is capable of opposing LDL's inductive effects and avoiding the ox-LDL's inhibition of eNOS. Paraoxonase 1 (PON1) is an HDL-associated enzyme esterase which appears to contribute to the antioxidant and antiatherosclerotic capabilities of HDL-C. “Healthy HDL,” namely the particle that contains the active Paraoxonase 1, has the power to suppress the formation of oxidized lipids. “Dysfunctional HDL,” on the contrary, has reduced Paraoxonase 1 enzyme activity and not only fails in its mission but also potentially leads to greater formation of oxidized lipids/lipoproteins to cause endothelial dysfunction. The association of HDL-C PON1 and endothelial dysfunction depends largely on the molecules with exact damaging effect on NO synthase coupling. Loss of nitric oxide bioavailability has a pivotal role in endothelial dysfunction preceding the appearance of atherosclerosis. Analyses of HDL-C and Paraoxonase1 would be more important in the diagnosis and treatment of atherosclerosis in the very near future.
PMCID: PMC3814057  PMID: 24222847
12.  Paraoxonase: Its antiatherogenic role in chronic renal failure 
Indian Journal of Nephrology  2010;20(1):9-14.
Paraoxonase (PON) is an aryldialkylphosphatase, which reversibly binds and hydrolyzes organophosphates. The PON family has three members (PON1, PON2 and PON3); they share structural properties and enzymatic activities. PON1 is shown to reside over high density lipoprotein (HDL) and has both antioxidant and antiatherogenic functions. Function of PON2 and PON3 are speculative and still under research. Several methodologies were developed over the years to determine the activity and mass of PON1, of which spectrophotometer-based methods using certain chemicals as substrate predominate. Several studies have shown decreased levels of PON1 in chronic renal failure (CRF) patients, particularly those on hemodialysis. The role of PON1 in development of cardiovascular disease has drawn considerable attention in recent years. Several authors have shown decreased levels of HDL and PON1 activity in CRF patients on hemodialysis and reported this to be a risk factor in the development of CVD. Enhancement or maintenance of the PON1 activity may prevent development of CVDs and its consequences in patients on hemodialysis.
PMCID: PMC2878404  PMID: 20535264
Atherosclerosis; chronic renal failure; hemodialysis; high density lipoprotein; low density lipoprotein oxidation; paraoxonase
13.  Lipid complex of apolipoprotein A-I mimetic peptide 4F is a novel platform for Paraoxonase-1 binding and enhancing its activity and stability 
High density lipoprotein (HDL) associated paraoxonase-1 (PON1) is crucial for the anti-oxidant, anti-inflammatory, and anti-atherogenic properties of HDL. Discoidal apolipoprotein (apo)A-I:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) complex has been shown to be the most effective in binding PON1, stabilizing it, and enhancing its lactonase and inhibitory activity of low density lipoprotein oxidation. Based on our earlier study demonstrating that apoA-I mimetic peptide 4F forms discoidal complex with 1,2-dimyristoyl-sn-glycero-3-phosphocholine, we hypothesized that lipid complexes of 4F would be able to bind PON1 and enhance its activity and stability. To test our hypothesis, we have expressed and purified a recombinant PON1 (rPON1) and studied its interaction with 4F:POPC complex. Our studies show significant increase, compared to the control, in the paraoxonase activity and stability of rPON1 in the presence of 4F:POPC complex. We propose that 4F:POPC complex is a novel platform for PON1 binding, increasing its stability, and enhancing its enzyme activity. We propose a structural model for the 4F:POPC:PON1 ternary complex that is consistent with our results and published observations.
PMCID: PMC3555693  PMID: 23261466
High density lipoprotein; Peptide; Amphipathic helix; Apolipoprotein A-I; Lipid; Paraoxonase-1
14.  The roles of PON1 and PON2 in cardiovascular disease and innate immunity 
Current opinion in lipidology  2009;20(4):10.1097/MOL.0b013e32832ca1ee.
Purpose of review
The paraoxonase (PON) gene family includes 3 members, PON1, PON2, and PON3. In vitro and mouse studies have demonstrated that all three PONs are athero-protective. Some but not all human epidemiologic studies have observed associations between PON gene polymorphisms and risk of cardiovascular disease (CVD). In this review, we summarize studies published within the past year elucidating involvement of PON1 and PON2 in oxidative stress, cardiovascular disease, and innate immune responses.
Recent findings
In a prospective study, the PON1 192QQ genotype and low PON1 activity were associated with increased systemic oxidative stress and increased risk for cardiovascular disease. PON1 expression protected against Pseudomonas aeruginosa lethality in Drosophila, suggesting that PON1 can interfere with quorum sensing in vivo. PON2 attenuated macrophage triglyceride accumulation via inhibition of diacylglycerol acyltransferase 1. Over-expression of PON2 protected against endoplasmic reticulum (ER) stress-induced apoptosis when the stress was induced by interference with protein modification but not when ER stress was induced by Ca ++ deregulation.
Both mouse and human studies have demonstrated the anti-oxidative and athero-protective effects of PON1. The mechanisms by which PON2 exerts its athero-protective effects are emerging. Large-scale epidemiologic studies are needed to further examine the relationship between PON2 genetic polymorphisms and risk for CVD. Elucidation of the physiological substrates of the PON proteins is of particular importance to further advance this field.
PMCID: PMC3869948  PMID: 19474728
Atherosclerosis; high density lipoprotein; paraoxonases; oxidative stress; quorum sensing
15.  Mildly oxidized LDL induces an increased apolipoprotein J/paraoxonase ratio. 
Journal of Clinical Investigation  1997;99(8):2005-2019.
We have examined the effects of mildly oxidized LDL and atherosclerosis on the levels of two proteins associated with HDL; apolipoprotein J (apoJ), and paraoxonase (PON). On an atherogenic diet, PON activity decreased by 52%, and apoJ levels increased 2.8-fold in fatty streak susceptible mice, C57BL/6J (BL/6), but not in fatty streak resistant mice, C3H/HeJ (C3H). Plasma PON activity was also significantly decreased, and apoJ levels were markedly increased in apolipoprotein E knockout mice on the chow diet, resulting in a 9.2-fold increase in the apoJ/PON ratio as compared to controls. Furthermore, a dramatic increase in the apoJ/PON ratio (over 100-fold) was observed in LDL receptor knockout mice when they were fed a 0.15%-cholesterol-enriched diet. Injection of mildly oxidized LDL (but not native LDL) into BL/6 mice (but not in C3H mice) on a chow diet resulted in a 59% decrease in PON activity (P < 0.01) and a 3.6-fold increase in apoJ levels (P < 0.01). When an acute phase reaction was induced in rabbits, or the rabbits were placed on an atherogenic diet, hepatic mRNA for apoJ was increased by 2.7-fold and 2.8-fold, respectively. Treatment of HepG2 cells in culture with mildly oxidized LDL (but not native LDL) resulted in reduced mRNA levels for PON (3.0-fold decrease) and increased mRNA levels for apoJ (2.0-fold increase). In normolipidemic patients with angiographically documented coronary artery disease who did not have diabetes and were not on lipid-lowering medication (n = 14), the total cholesterol/HDL cholesterol ratio was 3.1+/-0.9 as compared to 2.9+/-0.4 in the controls (n = 19). This difference was not statistically significant. In contrast, the apoJ/PON ratio was 3.0+/-0.4 in the patients compared to 0.72+/-0.2 in the controls (P < 0.009). In a subset of these normolipidemic patients (n = 5), the PON activity was low (48+/-6.6 versus 98+/-17 U/ml for controls; P < 0.009), despite similar normal HDL levels, and the HDL from these patients failed to protect against LDL oxidation in co-cultures of human artery wall cells. We conclude that: (a) mildly oxidized LDL can induce an increased apoJ/PON ratio, and (b) the apoJ/PON ratio may prove to be a better predictor of atherosclerosis than the total cholesterol/HDL cholesterol ratio.
PMCID: PMC508026  PMID: 9109446
16.  Beyond reduction of atherosclerosis: PON2 provides apoptosis resistance and stabilizes tumor cells 
Cell Death & Disease  2011;2(1):e112-.
Major contributors to atherosclerosis are oxidative damage and endoplasmic reticulum (ER) stress-induced apoptosis; both of which can be diminished by the anti-oxidative protein paraoxonase-2 (PON2). ER stress is also relevant to cancer and associated with anti-cancer treatment resistance. Hence, we addressed, for the first time, whether PON2 contributes to tumorigenesis and apoptotic escape. Intriguingly, we found that several human tumors upregulated PON2 and such overexpression provided resistance to different chemotherapeutics (imatinib, doxorubicine, staurosporine, or actinomycin) in cell culture models. This was reversed after PON2 knock-down. Remarkably, just deficiency of PON2 caused apoptosis of selective tumor cells per se, demonstrating a previously unanticipated oncogenic function. We found a dual mechanistic role. During ER stress, high PON2 levels lowered redox-triggered induction of pro-apoptotic CHOP particularly via the JNK pathway, which prevented mitochondrial cell death signaling. Apart from CHOP, PON2 also diminished intrinsic apoptosis as it prevented mitochondrial superoxide formation, cardiolipin peroxidation, cytochrome c release, and caspase activation. Ligand-stimulated apoptosis by TRAIL or TNFα remained unchanged. Finally, PON2 knock-down caused vast reactive oxygen species formation and stimulated JNK-triggered CHOP expression, but inhibition of JNK signaling did not prevent cell death, demonstrating the pleiotropic, dominating anti-oxidative effect of PON2. Therefore, targeting redox balance is powerful to induce selective tumor cell death and proposes PON2 as new putative anti-tumor candidate.
PMCID: PMC3077287  PMID: 21368884
paraoxonase; apoptosis; endoplasmic reticulum stress; mitochondria; oxidative stress; CHOP
17.  Antioxidant and Anti-Inflammatory Role of Paraoxonase 1: Implication in Arteriosclerosis Diseases 
Paraoxonase 1 (PON1) is a hydrolytic enzyme with wide range of substrates, and capability to protect against lipid oxidation. Despite of the large number of compounds that can be hydrolyzed by paraoxonase, the biologically relevant substrates are still not clearly determined. There is a massive in vitro and in vivo data to demonstrate the beneficial effects of PON1 in several atherosclerosis-related processes. The enzyme is primarily expressed in liver; however, it is also localized in other tissues. PON1 attracted significant interest as a protein that is responsible for the most of antioxidant properties of high-density lipoprotein (HDL). Several bioactive molecules such as dietary polyphenols, aspirin and its hydrolysis product salicylate, are known to stimulate PON1 transcription activation in mouse liver and HepG2 cell line. Studies on the activity, function, and genetic makeup have revealed a protective role of PON1. Some striking data were obtained in PON1 gene knockout and PON1 transgenic mouse models and in human studies. The goal of this review is to assess the current understanding of PON1 expression, enzymatic and antioxidant activity, and its atheroprotective effects. Results from in vivo and in vitro basic studies; and from human studies on the association of PON1 with coronary artery disease (CAD) and ischemic stroke will be discussed.
PMCID: PMC3503369  PMID: 23181222
Antioxidants; Atherosclerosis; Cardiovascular disease; Coronary artery disease; PON1
18.  Protectors or Traitors: The Roles of PON2 and PON3 in Atherosclerosis and Cancer 
Journal of Lipids  2012;2012:342806.
Cancer and atherosclerosis are major causes of death in western societies. Deregulated cell death is common to both diseases, with significant contribution of inflammatory processes and oxidative stress. These two form a vicious cycle and regulate cell death pathways in either direction. This raises interest in antioxidative systems. The human enzymes paraoxonase-2 (PON2) and PON3 are intracellular enzymes with established antioxidative effects and protective functions against atherosclerosis. Underlying molecular mechanisms, however, remained elusive until recently. Novel findings revealed that both enzymes locate to mitochondrial membranes where they interact with coenzyme Q10 and diminish oxidative stress. As a result, ROS-triggered mitochondrial apoptosis and cell death are reduced. From a cardiovascular standpoint, this is beneficial given that enhanced loss of vascular cells and macrophage death forms the basis for atherosclerotic plaque development. However, the same function has now been shown to raise chemotherapeutic resistance in several cancer cells. Intriguingly, PON2 as well as PON3 are frequently found upregulated in tumor samples. Here we review studies reporting PON2/PON3 deregulations in cancer, summarize most recent findings on their anti-oxidative and antiapoptotic mechanisms, and discuss how this could be used in putative future therapies to target atherosclerosis and cancer.
PMCID: PMC3361228  PMID: 22666600
19.  Repeated Developmental Exposure of Mice to Chlorpyrifos Oxon Is Associated with Paraoxonase 1 (PON1)-Modulated Effects on Cerebellar Gene Expression 
Toxicological Sciences  2011;123(1):155-169.
Microarray analysis was used to examine effects of repeated postnatal exposure to chlorpyrifos oxon (CPO) on gene expression in the cerebellum of genetically modified mice. The high-density lipoprotein-associated enzyme paraoxonase 1 (PON1) plays a significant role in the detoxication of CPO, which is present in exposures and generated from chlorpyrifos (CPF) in vivo following exposure. Two factors are important in modulating toxicity of CPO, the Q192R PON1 polymorphism and PON1 plasma level, which is low at birth and increases throughout postnatal development. Mice used in these studies included wild type (PON1+/+), PON1 knockout (PON1−/−), and two transgenic lines (tgHuPON1Q192, tgHuPON1R192) expressing either human PON1Q192 or PON1R192 on the PON1−/− background. PON1R192 hydrolyzes CPO more efficiently than PON1Q192. All four genotypes exposed to CPO (0.35 or 0.50 mg/kg/day) daily from postnatal day (PND) 4 to PND 21 showed significant differences in gene expression on PND 22 compared with controls. Pathway analysis and Gene Set Analysis revealed multiple pathways and gene sets significantly affected by CPO exposure, including genes involved in mitochondrial dysfunction, oxidative stress, neurotransmission, and nervous system development. Comparison between genotypes revealed specific genes, gene sets, and pathways differentially affected between tgHuPON1Q192 and tgHuPON1R192 mice and between PON1−/− and PON1+/+ mice following CPO exposure. Repeated CPO exposure also resulted in a dose-related decrease in brain acetylcholinesterase activity during postnatal development in PON1−/− and tgHuPON1Q192 mice but not in PON1+/+ or tgHuPON1R192 mice. These findings indicate that PON1 status plays a critical role in modulating the effects of neonatal CPO exposure in the developing brain.
PMCID: PMC3164442  PMID: 21673326
chlorpyrifos; chlorpyrifos oxon; paraoxonase; organophosphorus insecticides; microarrays; neurotoxicity
20.  Both Paraoxonase-1 Genotype and Activity Do Not Predict the Risk of Future Coronary Artery Disease; the EPIC-Norfolk Prospective Population Study 
PLoS ONE  2009;4(8):e6809.
Paraoxonase-1 (PON1) is an antioxidant enzyme, that resides on high-density lipoprotein (HDL). PON1-activity, is heavily influenced by the PON1-Q192R polymorphism. PON1 is considered to protect against atherosclerosis, but it is unclear whether this relation is independent of its carrier, HDL. In order to evaluate the atheroprotective potential of PON1, we assessed the relationships among PON1-genotype, PON1-activity and risk of future coronary artery disease (CAD), in a large prospective case-control study.
Methodology/Principal Findings
Cases (n = 1138) were apparently healthy men and women aged 45–79 years who developed fatal or nonfatal CAD during a mean follow-up of 6 years. Controls (n = 2237) were matched by age, sex and enrollment time. PON1-activity was similar in cases and controls (60.7±45.3 versus 62.6±45.8 U/L, p = 0.3) and correlated with HDL-cholesterol levels (r = 0.16, p<0.0001). The PON1-Q192R polymorphism had a profound impact on PON1-activity, but did not predict CAD risk (Odds Ratio [OR] per R allele 0.98[0.84–1.15], p = 0.8). Using conditional logistic regression, quartiles of PON1-activity showed a modest inverse relation with CAD risk (OR for the highest versus the lowest quartile 0.77[0.63–0.95], p = 0.01; p-trend = 0.06). PON1-activity adjusted for Q192R polymorphism correlated better with HDL-cholesterol (r = 0.26, p<0.0001) and more linearly predicted CAD risk (0.79[0.64–0.98], p = 0.03; p-trend = 0.008). However, these relationships were abolished after adjustment for HDL (particles-cholesterol-size) and apolipoproteinA-I (0.94[0.74–1.18], p-trend = 0.3).
This study, shows that PON1-activity inversely relates to CAD risk, but not independent of HDL, due to its close association with the HDL-particle. These data strongly suggest that a low PON1-activity is not a causal factor in atherogenesis.
PMCID: PMC2728540  PMID: 19710913
21.  Autoimmune-Mediated Reduction of High-Density Lipoprotein-Cholesterol and Paraoxonase-1 Activity in SLE-Prone gld Mice 
Arthritis and rheumatism  2011;63(1):201-211.
To characterize modifications of high-density lipoprotein (HDL) in autoimmune gld (generalized lymphoproliferative disorder) mice that may be relevant to premature atherosclerosis in systemic lupus erythematosus and assess their relationship to specific aspects of autoimmune disease.
HDL-cholesterol (HDL-C), apolipoprotein-A1 (ApoA1), paraoxonase-1 (PON1) activity, hepatic gene expression and HDL biogenesis were measured in ageing female gld and wild-type (WT) congenic mice. Autoantibodies, lymphoid organs and cytokines were analyzed by enzyme-linked immunosorbent assay, flow cytometry and multiplex assay respectively.
Plasma HDL-C, HDL-ApoA1 and HDL-associated PON1 activity were reduced in ageing gld mice in association with the development of autoimmunity independently of changes in hepatic ApoA1 and PON1 expression or HDL biogenesis. Hepatic induction of the acute phase reactant, serum amyloid A-1, resulted in its incorporation onto HDL in gld mice. Deletion of the lipid-sensitive receptor, G2A, in gld mice (G2A-/-gld) attenuated reductions in HDL-C and PON1 activity without altering hepatic ApoA1 and PON1 expression, HDL biogenesis or levels of acute phase pro-inflammatory cytokines. Plasma anti-ApoA1 autoantibodies were elevated in ageing gld mice commensurate with detectable increases in ApoA1 immune complexes. Autoantibodies were lower in ageing G2A-/-gld mice compared to gld mice and anti-ApoA1 autoantibody levels were significantly related to HDL-C concentration (r=-0.645, p<0.00004) and PON1 activity (r=-0.555, p<0.0007) amongst autoimmune gld and G2A-/-gld mice.
Autoantibodies against ApoA1 contribute to reducing HDL-C and PON1 activity in autoimmune gld mice independently of hepatic HDL biogenesis, suggesting that functional impairment and premature clearance of HDL immune complexes may be principal mechanisms involved.
PMCID: PMC3032585  PMID: 20882670
22.  Paraoxonase Polymorphisms, Haplotypes, and Enzyme Activity in Latino Mothers and Newborns 
Environmental Health Perspectives  2006;114(7):985-991.
Recent studies have demonstrated widespread pesticide exposures in pregnant women and in children. Plasma paraoxonase 1 (PON1) plays an important role in detoxification of various organophosphates. The goals of this study were to examine in the Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS) birth cohort of Latina mothers and their newborns living in the Salinas Valley, California, the frequencies of five PON1 polymorphisms in the coding region (192QR and 55LM) and the promoter region (−162AG, −909CG, and −108CT) and to determine their associations with PON1 plasma levels [phenylacetate arylesterase (AREase)] and enzyme activities of paraoxonase (POase) and chlorpyrifos oxonase (CPOase). Additionally, we report results of PON1 linkage analysis and estimate the predictive value of haplotypes for PON1 plasma levels. We found that PON1−909, PON1−108, and PON1192 had an equal frequency (0.5) of both alleles, whereas PON1−162 and PON155 had lower variant allele frequencies (0.2). Nearly complete linkage disequilibrium was observed among coding and promoter polymorphisms (p < 0.001), except PON1192 and PON1−162 (p > 0.4). Children’s PON1 plasma levels (AREase ranged from 4.3 to 110.7 U/mL) were 4-fold lower than their mothers’ (19.8 to 281.4 U/mL). POase and CPOase activities were approximately 3-fold lower in newborns than in mothers. The genetic contribution to PON1 enzyme variability was higher in newborns (R2 = 25.1% by genotype and 26.3% by haplotype) than in mothers (R2 = 8.1 and 8.8%, respectively). However, haplotypes and genotypes were comparable in predicting PON1 plasma levels in mothers and newborns. Most of the newborn children and some pregnant women in this Latino cohort may have elevated susceptibility to organophosphate toxicity because of their PON1192 genotype and low PON1 plasma levels.
PMCID: PMC1513322  PMID: 16835048
chlorpyrifos; cord blood; haplotypes; Latino cohort; linkage disequilibrium; organophosphate; paraoxonase 1 (PON1) genotype; paraoxonase activity; pesticides; PON1 polymorphisms; pregnancy
23.  Modulation of paraoxonases during infectious diseases and its potential impact on atherosclerosis 
The paraoxonase (PON) gene family includes three members, PON1, PON2 and PON3, aligned in tandem on chromosome 7 in humans and on chromosome 6 in mice. All PON proteins share considerable structural homology and have the capacity to protect cells from oxidative stress; therefore, they have been implicated in the pathogenesis of several inflammatory diseases, particularly atherosclerosis. The major goal of this review is to highlight the modulation of each of the PONs by infective (bacterial, viral and parasitic) agents, which may shed a light on the interaction between infectious diseases and PONs activities in order to effectively reduce the risk of developing atherosclerosis.
PMCID: PMC3457911  PMID: 22824324
Atherosclerosis; Paraoxonases; Oxidative stress; Infectious diseases
24.  Paraoxonase 1 Polymorphism and Prenatal Pesticide Exposure Associated with Adverse Cardiovascular Risk Profiles at School Age 
PLoS ONE  2012;7(5):e36830.
Prenatal environmental factors might influence the risk of developing cardiovascular disease later in life. The HDL-associated enzyme paraoxonase 1 (PON1) has anti-oxidative functions that may protect against atherosclerosis. It also hydrolyzes many substrates, including organophosphate pesticides. A common polymorphism, PON1 Q192R, affects both properties, but a potential interaction between PON1 genotype and pesticide exposure on cardiovascular risk factors has not been investigated. We explored if the PON1 Q192R genotype affects cardiovascular risk factors in school-age children prenatally exposed to pesticides.
Pregnant greenhouse-workers were categorized as high, medium, or not exposed to pesticides. Their children underwent a standardized examination at age 6-to-11 years, where blood pressure, skin folds, and other anthropometric parameters were measured. PON1-genotype was determined for 141 children (88 pesticide exposed and 53 unexposed). Serum was analyzed for insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein 3 (IGFBP3), insulin and leptin. Body fat percentage was calculated from skin fold thicknesses. BMI results were converted to age and sex specific Z-scores.
Prenatally pesticide exposed children carrying the PON1 192R-allele had higher abdominal circumference, body fat content, BMI Z-scores, blood pressure, and serum concentrations of leptin and IGF-I at school age than unexposed children. The effects were related to the prenatal exposure level. For children with the PON1 192QQ genotype, none of the variables was affected by prenatal pesticide exposure.
Our results indicate a gene-environment interaction between prenatal pesticide exposure and the PON1 gene. Only exposed children with the R-allele developed adverse cardiovascular risk profiles thought to be associated with the R-allele.
PMCID: PMC3352943  PMID: 22615820
25.  Relationship of PON1 192 and 55 gene polymorphisms to calcific valvular aortic stenosis 
Introduction and Objectives
Paraoxonases may exert anti-atherogenic action by reducing lipid peroxidation. Previous studies examined associations between polymorphisms in the paraoxonase 1 (PON1) gene and development of coronary artery disease (CAD), with inconsistent results. Given the similarities in clinical and pathophysiological risk factors of CAD and calcific aortic valve stenosis (CAVS), we postulated a link between PON1 alleles and CAVS progression.
We investigated the association between PON1 55 and 192 single nucleotide polymorphisms (SNPs), their enzyme activity, and CAVS progression assessed by aortic valve area and transvalvular peak velocity in 67 consecutive patients with moderate CAVS and 251 healthy controls.
PON1 paraoxonase activity was higher in CAVS patients (P<0.001). The PON1 genotype Q192R SNP (P=0.03) and variant allele (R192) (P=0.01) frequencies differed between CAVS patients and controls. Significant association existed between PON1 enzyme activity, phenotypic effects of PON1 192 genotype polymorphisms, and CAVS progression, but not between PON1 55 and high-density lipoprotein (P=0.44) or low-density lipoprotein cholesterol (P=0.12), between 192 genotype and high-density lipoprotein (P=0.24) or low-density lipoprotein cholesterol (P=0.52).
The PON1 genotype Q192R SNP has an important effect on CAVS disease progression. This study helps outline a genotype-phenotype relationship for PON1 in this unique population.
PMCID: PMC3371627  PMID: 22720202
Calcific aortic stenosis; polymorphism; paraoxonase; atherosclerosis; genetics; association

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