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Clement E Furlong - One of the best experts on this subject based on the ideXlab platform.
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Paraoxonase-1 (PON1) Status Analysis Using Non-Organophosphate Substrates.
Current protocols, 2021Co-Authors: Judit Marsillach, Rebecca J Richter, Lucio G Costa, Clement E FurlongAbstract:Human paraoxonase-1 (PON1) is a high-density lipoprotein-associated enzyme with antioxidant, anti-inflammatory, and antiapoptotic roles. The ability of PON1 to hydrolyze specific organophosphate (OP) compounds and prevent accumulation of oxidized lipids in lipoproteins has prompted a large number of studies investigating PON1's role in modulating toxicity and disease. Most of these studies, however, have only focused on PON1 single nucleotide polymorphism analyses and have ignored PON1 activity levels, arguably the most important parameter in determining protection against exposure and disease. We developed a two-substrate activity assay termed "PON1 status" that reveals both the functional PON1192 genotype and plasma PON1 activity levels. While our previous studies with PON1 status demonstrated that both PON1192 functional genotype and enzymatic activity levels obtained exclusively by determining PON1 status are required for a proper evaluation of PON1's role in modulating OP exposures and risk of disease, the original PON1 status assay requires the use of highly toxic OP metabolites. As many laboratories are not prepared to handle such toxic compounds and the associated waste generated, determination of PON1 status has been limited to rather few studies. Here, we describe a PON1 status protocol that uses non-OP substrates with a resolution equivalent to that of the original PON1 status approach. We have also included useful suggestions to ensure the assays can easily be carried out in any laboratory. The protocols described here will enable a proper examination of the risk of exposure or susceptibility to disease in PON1 epidemiological studies without the need to handle highly toxic substrates. © 2021 Wiley Periodicals LLC. Basic Protocol: Determining PON1 status using non-organophosphate substrates Support Protocol 1: Experimental pathlength determination Support Protocol 2: PON1 DNA genotyping for the Q192R (rs662) polymorphism.
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Paraoxonase 1 (PON1) as a genetic determinant of susceptibility to organophosphate toxicity
Toxicology, 2012Co-Authors: Lucio G Costa, Gennaro Giordano, Toby B Cole, Judit Marsillach, Clement E FurlongAbstract:a b s t r a c t Paraoxonase (PON1) is an A-esterase capable of hydrolyzing the active metabolites (oxons) of a number of organophosphorus (OP) insecticides such as parathion, diazinon and chlorpyrifos. PON1 activity is highest in liver and in plasma. Human PON1 displays two polymorphisms in the coding region (Q192R and L55M) and several polymorphisms in the promoter and the 3 � -UTR regions. The Q192R polymorphism imparts differential catalytic activity toward some OP substrates, while the polymorphism at position −108 (C/T) is the major contributor of differences in the levels of PON1 expression. Both contribute to determining an individual's PON1 "status". Animal studies have shown that PON1 is an important determinant of OP toxicity. Administration of exogenous PON1 to rats or mice protects them from the toxicity of specific OPs. PON1 knockout mice display a high sensitivity to the toxicity of diazoxon and chlorpyrifos oxon, but not of paraoxon. In vitro catalytic efficiencies of purified PON192 alloforms for hydrolysis of specific oxon substrates accurately predict the degree of in vivo protection afforded by each isoform. Evidence is slowly emerging that a low PON1 status may increase susceptibility to OP toxicity in humans. Low PON1 activity may also contribute to the developmental toxicity and neurotoxicity of OPs, as shown by animal and human studies. © 2012 Published by Elsevier Ireland Ltd.
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repeated developmental exposure of mice to chlorpyrifos oxon is associated with paraoxonase 1 PON1 modulated effects on cerebellar gene expression
Toxicological Sciences, 2011Co-Authors: Toby B Cole, Lucio G Costa, Richard P Beyer, Theo K Bammler, Sarah S Park, Federico M Farin, Clement E FurlongAbstract: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.
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Pharmacological and dietary modulators of paraoxonase 1 (PON1) activity and expression: the hunt goes on
Biochemical Pharmacology, 2010Co-Authors: Lucio G Costa, Gennaro Giordano, Clement E FurlongAbstract:Paraoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated enzyme displaying esterase and lactonase activity. PON1 hydrolyzes several organophosphorus (OP) insecticides and nerve agents, a number of exogenous and endogenous lactones, and metabolizes toxic oxidized lipids of low density lipoproteins (LDL) and HDL. As such, PON1 plays a relevant role in determining susceptibility to OP toxicity, cardiovascular diseases and several other diseases. Serum PON1 activity in a given population can vary by at least 40-fold. Most of this variation can be accounted for by genetic polymorphisms in the coding region (Q192R, L55M) and in the promoter region (T-108C). However, exogenous factors may also modulate PON1 activity and/or level of expression. This paper examines various factors that have been found to positively modulate PON1. Certain drugs (e.g. hypolipemic and anti-diabetic compounds), dietary factors (antioxidants, polyphenols), and life-style factors (moderate alcohol consumption) appear to increase PON1 activity. Given the relevance of PON1 in protecting from certain environmental exposure and from cardiovascular and other diseases, there is a need for further mechanistic, animal, and clinical research in this area, and for consideration of possible alternative strategies for increasing the levels and activity of PON1.
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Paraoxonase ( PON1 ) and Organophosphate Toxicity
The Paraoxonases: Their Role in Disease Development and Xenobiotic Metabolism, 2008Co-Authors: L. G. Costa, K L Jansen, T B Cole, Clement E FurlongAbstract:Paraoxonase (PON1) is a high density lipoprotein-associated enzyme capable of hydrolyzing multiple substrates, including several organophosphorus (OP) insecticides and nerve agents, oxidized lipids and a number of drugs or pro-drugs. Several polymor- phisms in the PON1 gene have been described, which have been shown to affect either the catalytic efficiency of hydrolysis or the expression level of the enzyme. Animal studies have shown that PON1 is an important determinant of the toxicity of certain OPs. Evidence for this was provided by cross-species comparisons, by administration of exogenous PON1 and by experiments in PON1 knockout and transgenic mice. Low PON1 plays also a role in the higher susceptibility of the young to OP toxicity. Recent findings also suggest that PON1 may modulate the toxicity resulting from exposure to mixtures of OP compounds
Michael Aviram - One of the best experts on this subject based on the ideXlab platform.
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Paraoxonase 1 (PON1) reduces macrophage inflammatory responses.
Atherosclerosis, 2013Co-Authors: Saar Aharoni, Michael Aviram, Bianca FuhrmanAbstract:Abstract Objectives Paraoxonase 1 (PON1) was suggested to play an anti-inflammatory role. In the present study we questioned whether PON1 has a direct impact on macrophage inflammatory responses, and the possible functional implications of such effects. Methods and results Ex-vivo studies were performed with bone marrow-derived macrophages (BMDM) harvested from C57BL/6 and human-PON1 transgenic (PON1-Tg) mice, and for the in vitro studies the J774.A1 macrophage-like cell line was used. Pro-inflammatory (M1) activation was induced by LPS and INFγ. The spontaneous and M1-induced TNFα and IL-6 secretion were significantly reduced in BMDM derived from PON1-Tg vs. C57BL/6 mice. In vitro, PON1 dose-dependently attenuated both the spontaneous and M1-induced TNFα and IL-6 secretion, and contributed to the anti-inflammatory activity of HDL. Functionally, PON1 attenuated M1-induced production of reactive oxygen species (ROS), phagocytosis, and necrotic macrophage death. PON1 anti-inflammatory activity was mediated, at least in part, via binding to SR-BI, but was independent of the enzyme catalytic activity or of cholesterol efflux stimulation, and did not involve binding to ABCA1. Conclusions The present study demonstrates, for the first time, that PON1 directly suppresses macrophage pro-inflammatory responses. These findings suggest that PON1 decreases sustained pro-inflammatory reactions, which subsequently can attenuate plaque progression.
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the antioxidant hdl associated paraoxonase 1 PON1 attenuates diabetes development and stimulates β cell insulin release
Atherosclerosis, 2011Co-Authors: Marie Korengluzer, Michael Aviram, Edna Meilin, Tony HayekAbstract:Abstract Objective To analyze the direct effects of paraoxonase-1 (PON1) on diabetes development and on β-cell insulin release. Methods and results Injection of rePON1 to mice, prior to STZ-induced diabetes, resulted in reduced incidence of diabetes, as well as, in higher serum insulin levels. Incubation of β-cells with PON1 also dose-dependently increased insulin secretion and its cellular content. PON1 increased cell survival under high glucose levels, but not under high STZ concentrations. The addition of the PON1 carrier in the circulation – HDL, to βTC3 cell line, had an additive effect on PON1-induced insulin secretion. PON1 administration to mice or incubation with β-cells was associated with a substantial decreased oxidative stress. Just like PON1, the dietary anti-oxidants, pomegranate juice, punicalagin (major polyphenol in pomegranate) or vitamin E, also increased insulin release from βTC3, but unlike PON1, failed to increase insulin cellular content, suggesting a possible role for PON1 in insulin biosynthesis, separately from PON1 antioxidative effect. Both, PON1 catalytic activity and PON1 association to HDL, were not required for PON1 stimulation of insulin release from β-cells. However, the PON1 free sulfhydryl group was shown to be essential for insulin release by PON1, as blocking the PON1 SH group, abolished PON1 stimulatory effect on insulin secretion. Conclusion PON1 is a potent anti-diabetic enzyme that exerts this protection against diabetes through its antioxidative, as well as via its insulin stimulation properties on β-cells.
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Human carotid lesion linoleic acid hydroperoxide inhibits paraoxonase 1 (PON1) activity via reaction with PON1 free sulfhydryl cysteine 284
Free radical biology & medicine, 2010Co-Authors: Hagai Tavori, Michael Aviram, Soliman Khatib, Ramadan Musa, Dalit Mannheim, Ron Karmeli, Jacob VayaAbstract:Paraoxonase 1 (PON1) is an HDL-associated lactonase with antiatherogenic properties. These include dampening the oxidation properties of human carotid lesion lipid extract (LLE), which in turn inactivates the enzyme. The aims of this study were to identify the PON1 inhibitor in LLE and explore the mechanism of inhibition. LLE inhibited both recombinant PON1 and HDL-PON1 lactonase activity in a dose- and time-dependent manner. Addition of antioxidants or electrophiles to LLE did not prevent PON1 inhibition. LLE was unable to inhibit a PON1 mutant lacking Cys284, whereas it did inhibit all other PON1 mutants tested. The inhibitor in the LLE was identified as linoleic acid hydroperoxide (LA-OOH) and inhibition was specific to this hydroperoxide. During its inhibition, PON1 acted like a peroxidase enzyme, reducing LA-OOH to LA-hydroxide via its Cys284. A similar reaction occurred with external thiols, such as DDT or cysteine, which also prevented PON1 inhibition and restored enzyme activity after inhibition. Thus, the antiatherogenic properties of HDL could be, at least in part, related to the sulfhydryl-reducing characteristics of its associated PON1, which are further protected and recycled by the sulfhydryl amino acid cysteine.
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paraoxonase 1 PON1 expression in hepatocytes is upregulated by pomegranate polyphenols a role for ppar γ pathway
Atherosclerosis, 2010Co-Authors: Jasmin Khateeb, Michael Aviram, Anna Gantman, Adam J Kreitenberg, Bianca FuhrmanAbstract:Abstract Objective Serum paraoxonase-1 (PON1) expression is regulated by polyphenols, shown to activate the peroxisome proliferator-activated receptor γ (PPARγ). Pomegranate juice (PJ) is a polyphenol-rich fruit. Because promoter sequence of PON1 gene indicates that it could be regulated by nuclear receptors, we investigated the effect of PJ polyphenols on PON1 gene expression in HuH7 hepatocytes. Methods and results PON1 protein or mRNA expression, determined by immunocytochemistry, or quantitative PCR, respectively, as well as PON1 gene promoter activation, was significantly increased in hepatocytes incubated with PJ or with its major polyphenols punicalagin, or gallic acid (GA). Ellagic acid (EA) elicited only modest stimulatory effect. Accordingly, PJ, punicalagin, GA, and less so EA, dose-dependently increased cell-associated and hepatocyte-secreted PON1 arylesterase activity. Functionally, the secreted PON1 exhibited biological activity by protecting LDL and HDL from oxidation. Finally, PJ polyphenols upregulated the hepatocyte PON1 expression, at least in part, via the intracellular signaling cascade PPARγ-PKA-cAMP. Conclusions This study shows for the first time that PJ polyphenols have a specific transcriptional role in hepatocyte PON1 expression upregulation, and its secretion to the medium. We conclude that the anti-atherogenic characteristics of PJ polyphenols are modulated, at least in part, via hepatocyte PON1 upregulation and its subsequent release to the medium.
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Macrophage paraoxonase 1 (PON1) binding sites.
Biochemical and biophysical research communications, 2008Co-Authors: Michal Efrat, Michael AviramAbstract:Abstract Paraoxonase 1 (PON1), an HDL-associated esterase, is known to possess anti-oxidant and anti-atherogenic properties. PON1 was shown to protect macrophages from oxidative stress, to inhibit macrophage cholesterol biosynthesis, and to stimulate HDL-mediated cholesterol efflux from the cells. The aim of the present study was to characterize macrophage PON1 binding sites which could be responsible for the above anti-atherogenic activities. Incubation of FITC-labeled recombinant PON1 with J774 A.1 macrophage-like cell line at 37 °C, resulted in cellular binding and internalization of PON1, leading to PON1 localization in the cell’s cytoplasm compartment. In order to determine whether PON1 uptake is mediated via a specific binding to the macrophage, FITC-labeled recombinant PON1 was incubated with macrophages at 4 °C, followed by cell membranes separation. Macrophage membrane fluorescence was shown to be directly and dose-dependently related to the labeled PON1 concentration. Furthermore, binding assays performed at 4 and at 37 °C, using labeled and non-labeled recombinant PON1 (for competitive inhibition), demonstrated a dose-dependent significant 30% decrement in labeled PON1 binding to the macrophages, by the non-labeled PON1. Similarly, binding assays, using labeled PON1 and non-labeled HDL (the natural carrier of PON1 in the circulation) indicated that HDL decreased the binding of labeled PON1 to macrophages by 25%. Unlike HDL, LDL had no effect on labeled PON1 binding to macrophages. Finally, HDL were pre incubated without or with PON1 or apolipoprotein AI (apoAI) antibodies, in order to block PON1 or apoAI ability to bind to the cells. HDL incubation with antibody to PON1 or to apoAI significantly decreased HDL ability to inhibit macrophages-mediated LDL oxidation (by 32% or by 25%, respectively). A similar trend was also observed for HDL-mediated cholesterol efflux from macrophages, with an inhibitory effect of 35% or 19%, respectively. These results suggest that blocking HDL binding to macrophages through its apo A-I, and more so, via its PON1, results in the attenuation of HDL-PON1 biological activities. In conclusion, PON1 specifically binds to macrophage binding sites, leading to anti-atherogenic effects. Macrophage PON1 binding sites may thus be a target for future cardio protection therapy.
Lucio G Costa - One of the best experts on this subject based on the ideXlab platform.
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Paraoxonase-1 (PON1) Status Analysis Using Non-Organophosphate Substrates.
Current protocols, 2021Co-Authors: Judit Marsillach, Rebecca J Richter, Lucio G Costa, Clement E FurlongAbstract:Human paraoxonase-1 (PON1) is a high-density lipoprotein-associated enzyme with antioxidant, anti-inflammatory, and antiapoptotic roles. The ability of PON1 to hydrolyze specific organophosphate (OP) compounds and prevent accumulation of oxidized lipids in lipoproteins has prompted a large number of studies investigating PON1's role in modulating toxicity and disease. Most of these studies, however, have only focused on PON1 single nucleotide polymorphism analyses and have ignored PON1 activity levels, arguably the most important parameter in determining protection against exposure and disease. We developed a two-substrate activity assay termed "PON1 status" that reveals both the functional PON1192 genotype and plasma PON1 activity levels. While our previous studies with PON1 status demonstrated that both PON1192 functional genotype and enzymatic activity levels obtained exclusively by determining PON1 status are required for a proper evaluation of PON1's role in modulating OP exposures and risk of disease, the original PON1 status assay requires the use of highly toxic OP metabolites. As many laboratories are not prepared to handle such toxic compounds and the associated waste generated, determination of PON1 status has been limited to rather few studies. Here, we describe a PON1 status protocol that uses non-OP substrates with a resolution equivalent to that of the original PON1 status approach. We have also included useful suggestions to ensure the assays can easily be carried out in any laboratory. The protocols described here will enable a proper examination of the risk of exposure or susceptibility to disease in PON1 epidemiological studies without the need to handle highly toxic substrates. © 2021 Wiley Periodicals LLC. Basic Protocol: Determining PON1 status using non-organophosphate substrates Support Protocol 1: Experimental pathlength determination Support Protocol 2: PON1 DNA genotyping for the Q192R (rs662) polymorphism.
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Paraoxonase 1 (PON1) as a genetic determinant of susceptibility to organophosphate toxicity
Toxicology, 2012Co-Authors: Lucio G Costa, Gennaro Giordano, Toby B Cole, Judit Marsillach, Clement E FurlongAbstract:a b s t r a c t Paraoxonase (PON1) is an A-esterase capable of hydrolyzing the active metabolites (oxons) of a number of organophosphorus (OP) insecticides such as parathion, diazinon and chlorpyrifos. PON1 activity is highest in liver and in plasma. Human PON1 displays two polymorphisms in the coding region (Q192R and L55M) and several polymorphisms in the promoter and the 3 � -UTR regions. The Q192R polymorphism imparts differential catalytic activity toward some OP substrates, while the polymorphism at position −108 (C/T) is the major contributor of differences in the levels of PON1 expression. Both contribute to determining an individual's PON1 "status". Animal studies have shown that PON1 is an important determinant of OP toxicity. Administration of exogenous PON1 to rats or mice protects them from the toxicity of specific OPs. PON1 knockout mice display a high sensitivity to the toxicity of diazoxon and chlorpyrifos oxon, but not of paraoxon. In vitro catalytic efficiencies of purified PON192 alloforms for hydrolysis of specific oxon substrates accurately predict the degree of in vivo protection afforded by each isoform. Evidence is slowly emerging that a low PON1 status may increase susceptibility to OP toxicity in humans. Low PON1 activity may also contribute to the developmental toxicity and neurotoxicity of OPs, as shown by animal and human studies. © 2012 Published by Elsevier Ireland Ltd.
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repeated developmental exposure of mice to chlorpyrifos oxon is associated with paraoxonase 1 PON1 modulated effects on cerebellar gene expression
Toxicological Sciences, 2011Co-Authors: Toby B Cole, Lucio G Costa, Richard P Beyer, Theo K Bammler, Sarah S Park, Federico M Farin, Clement E FurlongAbstract: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.
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Pharmacological and dietary modulators of paraoxonase 1 (PON1) activity and expression: the hunt goes on
Biochemical Pharmacology, 2010Co-Authors: Lucio G Costa, Gennaro Giordano, Clement E FurlongAbstract:Paraoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated enzyme displaying esterase and lactonase activity. PON1 hydrolyzes several organophosphorus (OP) insecticides and nerve agents, a number of exogenous and endogenous lactones, and metabolizes toxic oxidized lipids of low density lipoproteins (LDL) and HDL. As such, PON1 plays a relevant role in determining susceptibility to OP toxicity, cardiovascular diseases and several other diseases. Serum PON1 activity in a given population can vary by at least 40-fold. Most of this variation can be accounted for by genetic polymorphisms in the coding region (Q192R, L55M) and in the promoter region (T-108C). However, exogenous factors may also modulate PON1 activity and/or level of expression. This paper examines various factors that have been found to positively modulate PON1. Certain drugs (e.g. hypolipemic and anti-diabetic compounds), dietary factors (antioxidants, polyphenols), and life-style factors (moderate alcohol consumption) appear to increase PON1 activity. Given the relevance of PON1 in protecting from certain environmental exposure and from cardiovascular and other diseases, there is a need for further mechanistic, animal, and clinical research in this area, and for consideration of possible alternative strategies for increasing the levels and activity of PON1.
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The Functional Consequences of Polymorphisms in the Human PON1 Gene
The Paraoxonases: Their Role in Disease Development and Xenobiotic Metabolism, 2008Co-Authors: Clement E Furlong, Lucio G Costa, Rebecca J Richter, Victoria H. Brophy, Christopher S. Carlson, Mark J. Rieder, Deborah A. Nickerson, J. Ranchalis, Aldon J. LusisAbstract:Early research on population distributions of plasma PON1 paraoxonase activity indicated a polymorphic distribution with high, intermediate and low metabolizers. Cloning and characterization of the cDNA encoding human PON1 and follow-on experiments demonstrated that the molecular basis of the activity polymorphism (PM) was a Q192R PM with PON1R192 specifying high paraoxonase activity. Further research demonstrated that the PON1192 polymorphism had little effect on the catalytic efficiencies of hydrolysis of phenylacetate and diazoxon (DZO), but did affect the efficiencies of hydrolysis of chlorpyrifos oxon (CPO), soman and sarin, with PON1R192 having a higher efficiency of CPO hydrolysis and PON1Q192 having higher rates of hydrolysis of soman and sarin. Plots of rates of DZO hydrolysis (at a salt concentration that differentially inhibited PON1R192) vs. paraoxon hydrolysis clearly separated the three PON1192 phenotypes (QQ, QR, RR) and also showed a wide range of activity among individuals with the same PON1192 genotype. The term PON1 status was introduced to include both PON1192 functional genotype and plasma PON1 level,both important in determining risk for either exposure to specific organophosphorus compounds (OPs) or disease. Characterization of 5 promoter-region polymorphisms by several groups indicated that an Sp1 binding site was responsible for significant(~30%) variation in plasma PON1 levels. Re-sequencing of the PON1 genes of 47 individuals (24 African-American/23 European) revealed an additional 180 polymorphisms in 27 kb of the PON1 genomic DNA including 8 more 5' regulatory region PMs, 1 coding region polymorphism (W194X), 162 additional intronic PMs and 9 additional 3' UTR PMs. The generation of PON1 null mice and “PON1 humanized mice” expressing either tgHuPON1R192 or tgHuPON1Q192 at the same levels on the PON1−/− background allowed for a functional analysis of the Q192R PM under physiological conditions. Toxicology experiments with the PON1 humanized mice and the PON1 null mice injected with purified human PON1192 alloforms clearly demonstrated that the catalytic efficiency of substrate hydrolysis is important in determining whether PON1 is able to protect against a given OP exposure. HuPON1R192 protects well against CPO and DZO exposure, but HuPON1Q192 does not protect well against CPO exposure and neither protects against PO exposure. Studies on PON1 status and carotid artery disease show that low PON1 levels are a risk factor. The effects of PON1192 alloforms on rates of hydrolysis of quorum sensing factors are not yet known. Taken together, these data along with those of the leading researchers in the PON1 field indicate that it is important to measure PON1 levels/activities in any epidemiological study. SNP analysis alone is inadequate for epidemiological studies, due to the wide variability of PON1 levels within the three PON1192 genotypes Q/Q, Q/R R/R). Even the most comprehensive PON1 SNP analyses are unable to accurately predict PON1 levels. PON1 activity or level accurately predicts CHD risk, while genotype does not
Rebecca J Richter - One of the best experts on this subject based on the ideXlab platform.
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Paraoxonase-1 (PON1) Status Analysis Using Non-Organophosphate Substrates.
Current protocols, 2021Co-Authors: Judit Marsillach, Rebecca J Richter, Lucio G Costa, Clement E FurlongAbstract:Human paraoxonase-1 (PON1) is a high-density lipoprotein-associated enzyme with antioxidant, anti-inflammatory, and antiapoptotic roles. The ability of PON1 to hydrolyze specific organophosphate (OP) compounds and prevent accumulation of oxidized lipids in lipoproteins has prompted a large number of studies investigating PON1's role in modulating toxicity and disease. Most of these studies, however, have only focused on PON1 single nucleotide polymorphism analyses and have ignored PON1 activity levels, arguably the most important parameter in determining protection against exposure and disease. We developed a two-substrate activity assay termed "PON1 status" that reveals both the functional PON1192 genotype and plasma PON1 activity levels. While our previous studies with PON1 status demonstrated that both PON1192 functional genotype and enzymatic activity levels obtained exclusively by determining PON1 status are required for a proper evaluation of PON1's role in modulating OP exposures and risk of disease, the original PON1 status assay requires the use of highly toxic OP metabolites. As many laboratories are not prepared to handle such toxic compounds and the associated waste generated, determination of PON1 status has been limited to rather few studies. Here, we describe a PON1 status protocol that uses non-OP substrates with a resolution equivalent to that of the original PON1 status approach. We have also included useful suggestions to ensure the assays can easily be carried out in any laboratory. The protocols described here will enable a proper examination of the risk of exposure or susceptibility to disease in PON1 epidemiological studies without the need to handle highly toxic substrates. © 2021 Wiley Periodicals LLC. Basic Protocol: Determining PON1 status using non-organophosphate substrates Support Protocol 1: Experimental pathlength determination Support Protocol 2: PON1 DNA genotyping for the Q192R (rs662) polymorphism.
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The Functional Consequences of Polymorphisms in the Human PON1 Gene
The Paraoxonases: Their Role in Disease Development and Xenobiotic Metabolism, 2008Co-Authors: Clement E Furlong, Lucio G Costa, Rebecca J Richter, Victoria H. Brophy, Christopher S. Carlson, Mark J. Rieder, Deborah A. Nickerson, J. Ranchalis, Aldon J. LusisAbstract:Early research on population distributions of plasma PON1 paraoxonase activity indicated a polymorphic distribution with high, intermediate and low metabolizers. Cloning and characterization of the cDNA encoding human PON1 and follow-on experiments demonstrated that the molecular basis of the activity polymorphism (PM) was a Q192R PM with PON1R192 specifying high paraoxonase activity. Further research demonstrated that the PON1192 polymorphism had little effect on the catalytic efficiencies of hydrolysis of phenylacetate and diazoxon (DZO), but did affect the efficiencies of hydrolysis of chlorpyrifos oxon (CPO), soman and sarin, with PON1R192 having a higher efficiency of CPO hydrolysis and PON1Q192 having higher rates of hydrolysis of soman and sarin. Plots of rates of DZO hydrolysis (at a salt concentration that differentially inhibited PON1R192) vs. paraoxon hydrolysis clearly separated the three PON1192 phenotypes (QQ, QR, RR) and also showed a wide range of activity among individuals with the same PON1192 genotype. The term PON1 status was introduced to include both PON1192 functional genotype and plasma PON1 level,both important in determining risk for either exposure to specific organophosphorus compounds (OPs) or disease. Characterization of 5 promoter-region polymorphisms by several groups indicated that an Sp1 binding site was responsible for significant(~30%) variation in plasma PON1 levels. Re-sequencing of the PON1 genes of 47 individuals (24 African-American/23 European) revealed an additional 180 polymorphisms in 27 kb of the PON1 genomic DNA including 8 more 5' regulatory region PMs, 1 coding region polymorphism (W194X), 162 additional intronic PMs and 9 additional 3' UTR PMs. The generation of PON1 null mice and “PON1 humanized mice” expressing either tgHuPON1R192 or tgHuPON1Q192 at the same levels on the PON1−/− background allowed for a functional analysis of the Q192R PM under physiological conditions. Toxicology experiments with the PON1 humanized mice and the PON1 null mice injected with purified human PON1192 alloforms clearly demonstrated that the catalytic efficiency of substrate hydrolysis is important in determining whether PON1 is able to protect against a given OP exposure. HuPON1R192 protects well against CPO and DZO exposure, but HuPON1Q192 does not protect well against CPO exposure and neither protects against PO exposure. Studies on PON1 status and carotid artery disease show that low PON1 levels are a risk factor. The effects of PON1192 alloforms on rates of hydrolysis of quorum sensing factors are not yet known. Taken together, these data along with those of the leading researchers in the PON1 field indicate that it is important to measure PON1 levels/activities in any epidemiological study. SNP analysis alone is inadequate for epidemiological studies, due to the wide variability of PON1 levels within the three PON1192 genotypes Q/Q, Q/R R/R). Even the most comprehensive PON1 SNP analyses are unable to accurately predict PON1 levels. PON1 activity or level accurately predicts CHD risk, while genotype does not
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PON1 status of farmworker mothers and children as a predictor of organophosphate sensitivity
Pharmacogenetics and Genomics, 2006Co-Authors: Clement E Furlong, Rebecca J Richter, Nina Holland, Asa Bradman, Brenda EskenaziAbstract:The objective was to determine PON1 status as a predictor for organophosphorus insecticide sensitivity in a cohort of Latina mothers and newborns from the Salinas Valley, California, an area with high levels of organophosphorus insecticide use. PON1 status was established for 130 pregnant Latina women and their newborns using a high-throughput two substrate activity/analysis method which plots rates of diazoxon (DZO) hydrolysis against rates of paraoxon (PO) hydrolysis. Arylesterase activity (AREase) was determined using phenylacetate as a substrate, allowing comparison of PON1 levels across PON1192 genotypes in mothers and children. Phenylacetate hydrolysis is not affected by the Q192R polymorphism. Among newborns, levels of PON1 (AREase) varied by 26-fold (4.3-110.7 U/ml) and among mothers by 14-fold (19.8-281.4 U/ml). On average, children's PON1 levels were four-fold lower than the mothers' PON1 levels (P<0.001). Average PON1 levels in newborns were comparable with reported hPON1 levels in transgenic mice expressing human PON1Q192 or PON1R192, allowing for prediction of relative sensitivity to chlorpyrifos oxon (CPO) and DZO. The predicted range of variability in sensitivity of mothers and children in the same Latino cohort was 65-fold for DZO and 131 to 164-fold for CPO. Overall, these findings indicate that many of the newborns and some of the mothers in this cohort would be more susceptible to the adverse effects of specific organophosphorus pesticide exposure due to their PON1 status. Of particular concern are exposures of pregnant mothers and newborns with low PON1 status.
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the correlation of paraoxonase PON1 activity with lipid and lipoprotein levels differs with vascular disease status
Journal of Lipid Research, 2005Co-Authors: Rebecca J Richter, Laura A. Mckinstry, Laura S Rozek, Thomas S Hatsukami, Jane E Ranchalis, Karen Nakayama, David A Gortner, Edward J Boyko, Gerard D SchellenbergAbstract:Paraoxonase (PON1) is an HDL-associated en- zyme. Low PON1 activity predicts vascular disease status and is a more reliable predictor of vascular disease than are func- tional PON1 genotypes. There is evidence that the relation- ship of PON1 to vascular disease is, in part, due to its anti- oxidant activity. However, the physical relationship of PON1 with HDL and the existence of cholesterol pathway regula- tory elements at the PON1 locus suggest a further relation- ship of PON1 with lipoproteins, which may contribute to its role in vascular disease. We investigated the relationship of PON1 activity and genotype to lipid-related traits in 91 Cau- casian men with severe carotid artery disease and 184 with- out vascular disease who were not on lipid-lowering medica- tions. Prior studies of PON1 relationship to lipids have not stratified by disease status. We found that PON1 activity was correlated with HDL traits in controls and with LDL- and VLDL-related traits in cases. We hypothesize differences in the joint regulation of PON1 and lipoproteins in cases and controls. —Rozek, L. S., T. S. Hatsukami, R. J. Richter, J. Ranchalis, K. Nakayama, L. A. McKinstry, D. A. Gortner, E. Boyko, G. D. Schellenberg, C. E. Furlong, and G. P. Jarvik. The correlation of paraoxonase (PON1) activity with lipid and lipoprotein levels differs with vascular disease status. J. Lipid Res. 2005. 46: 1888-1895.
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role of paraoxonase PON1 status in pesticide sensitivity genetic and temporal determinants
Neurotoxicology, 2005Co-Authors: Clement E Furlong, Diana M Shih, Toby B Cole, Gail P Jarvik, Christina Pettanbrewer, Gary K Geiss, Rebecca J Richter, Aaron D Tward, Aldons J Lusis, Lucio G CostaAbstract:Individual differences in detoxication capacities for specific organophosphorous (OP) compounds are due largely to differences in catalytic efficiency or abundance of the HDL-associated enzyme, paraoxonase (PONI). First, we provide evidence that children less than 2 years of age represent a particularly susceptible population for OP exposure due to low abundance of PON1 and variable onset of plasma PONI activity. Second, we describe studies examining the neurotoxic effects of chronic, low-level OP pesticide exposure in mice. PON1 knockout (PON1 - / - ) and wild-type mice were exposed chronically (PN4 to PN21) to low levels of chlorpyrifos oxon (CPO). Endpoints included cholinesterase activity, histopathology, gene expression, and behavior. Even at PN4, when PON1 levels were low in wild-type mice, PON1 - / - mice were more sensitive to inhibition of brain cholinesterase by CPO. At PN22, and persisting as long as 4 months, chronic developmental exposure to 0.18 mg/kg/d or 0.25 mg/kg/d CPO resulted in perinuclear vacuolization of cells in a discrete area of the neocortex and irregular distribution of neurons in the cortical plate, with an increase in the number of affected cells at 0.25 mg/kg/d. Third, we describe a transgenic mouse model in which human transgenes encoding either hPON1 Q 1 9 2 or hPON1 R 1 9 2 were expressed at equal levels in place of mouse PON1. The developmental onset ofexpression followed the mouse time course and was identical for the two transgenes, allowing these mice to be used to assess the importance of the Q192R polymorphism during development. Adult mice expressing hPON1 R 1 9 2 were significantly more resistant than hPON1 Q 1 9 2 mice to CPO toxicity. Our studies indicate that children less than 2 years old, especially those homozygous for PON1 Q 1 9 2 , would be predicted to be particularly susceptible to CPO toxicity.
Diana M Shih - One of the best experts on this subject based on the ideXlab platform.
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Suppression of inflammatory arthritis in human serum paraoxonase 1 transgenic mice
Scientific Reports, 2020Co-Authors: Christina Charles-schoeman, Diana M Shih, Jennifer Wang, Ani Shahbazian, Yuen Yin Lee, Xiaoyan Wang, Victor Grijalva, Ernest Brahn, Asokan Devarajan, Christy MontanoAbstract:Paraoxonase 1(PON1) is an HDL-associated protein, which metabolizes inflammatory, oxidized lipids associated with atherosclerotic plaque development. Because oxidized lipid mediators have also been implicated in the pathogenesis of rheumatoid arthritis (RA), we evaluated the role of PON1 in murine inflammatory arthritis. K/BxN serum transfer (STIA) or collagen antibody transfer (CAIA) was used for arthritis induction in B6 mice homozygous for the PON1 human transgene [PON1Tg], PON1 knock-out mice [PON1KO], and wild type littermate control mice [WT]. Experiments were also performed in K/BxN mice with chronic arthritis, and in RA patients and healthy controls. Arthritis activity in K/BxN mice was associated with a marked dyslipidemia, lower PON1 activity and higher bioactive lipid mediators (BLM), as well as a dysregulated hepatic lipid gene expression profile. Higher serum PON1 activity correlated with lower BLM and lower arthritis activity in both K/BxN mice and RA patients. Overexpression of the human PON1 transgene was associated with reduced inflammatory arthritis, which correlated strongly with higher circulating PON1 activity, upregulation of the hepatic glutathione pathway, and reduction of circulating BLM. These results implicate PON1 as a potential novel therapeutic target for joint disease in RA with potential for vascular benefit, which warrants further investigation.
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metabolism and neurotoxicity of homocysteine thiolactone in mice evidence for a protective role of paraoxonase 1
Journal of Alzheimer's Disease, 2012Co-Authors: Kamila Borowczyk, Diana M Shih, Hieronim JakubowskiAbstract:Homocysteine (Hcy)-thiolactone is toxic, induces epileptic seizures in rodents, and has been implicated in Alzheimer’s disease. Paraoxonase 1 (PON1), a component of high-density lipoprotein, hydrolyzes Hcy-thiolactone in vitro. Whether this reflects a physiological function and whether PON1 can protect against Hcy-thiolactone toxicity was unknown. Here we show that Hcy-thiolactone was elevated in brains of PON1−/− mice (1.5-fold, p = 0.047) and that PON1−/− mice excrete more Hcy-thiolactone than wild type animals (2.4-fold, p = 0.047). The frequency of seizures induced by intraperitoneal injections of L-Hcy-thiolactone was significantly higher in PON1−/− mice compared with wild type animals (52.8% versus 29.5%, p = 0.042); the latency of seizures was lower in PON1−/− mice than in wild type animals (31.8 min versus 41.2 min, p = 0.019). Using the PON1 null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself is neurotoxic in vivo. Our findings indicate that PON1 protects mice against Hcy-thiolactone neurotoxicity by hydrolyzing it in the brain, and suggest a mechanism by which PON1 can protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer’s disease.
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trypanosoma congolense paraoxonase 1 prolongs survival of infected mice
Experimental Parasitology, 2006Co-Authors: Kum Kum S Bhasin, Diana M Shih, Aaron D Tward, David A Campbell, Aldons J LusisAbstract:In vitro studies have suggested that a fraction of human high density lipoprotein (HDL), termed trypanosome lysis factor (TLF), can protect against trypanosome infection. We examined the involvement of two proteins located in the TLF fraction, apolipoprotein A-II (apoA-II) and paraoxonase 1 (PON1), against trypanosome infection. To test whether PON1 is involved in trypanosome resistance, we infected human PON1 transgenic mice, PON1 knockout mice, and wild-type mice with Trypanosoma congolense. When challenged with the same dosage of trypanosomes, mice overexpressing PON1 lived significantly longer than wild-type mice, and mice deficient in PON1 lived significantly shorter. In contrast, mice overexpressing another HDL associated protein, apoA-II, had the same survival as wild-type mice. Together, these data suggest that PON1 provides protection against trypanosome infection. In vitro studies using T. brucei brucei indicated that HDL particles containing PON1 and those depleted of PON1 did not differ in their lysis ability, suggesting that protection by PON1 is indirect. Our data are consistent with an in vivo role of HDL protection against trypanosome infection.
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role of paraoxonase PON1 status in pesticide sensitivity genetic and temporal determinants
Neurotoxicology, 2005Co-Authors: Clement E Furlong, Diana M Shih, Toby B Cole, Gail P Jarvik, Christina Pettanbrewer, Gary K Geiss, Rebecca J Richter, Aaron D Tward, Aldons J Lusis, Lucio G CostaAbstract:Individual differences in detoxication capacities for specific organophosphorous (OP) compounds are due largely to differences in catalytic efficiency or abundance of the HDL-associated enzyme, paraoxonase (PONI). First, we provide evidence that children less than 2 years of age represent a particularly susceptible population for OP exposure due to low abundance of PON1 and variable onset of plasma PONI activity. Second, we describe studies examining the neurotoxic effects of chronic, low-level OP pesticide exposure in mice. PON1 knockout (PON1 - / - ) and wild-type mice were exposed chronically (PN4 to PN21) to low levels of chlorpyrifos oxon (CPO). Endpoints included cholinesterase activity, histopathology, gene expression, and behavior. Even at PN4, when PON1 levels were low in wild-type mice, PON1 - / - mice were more sensitive to inhibition of brain cholinesterase by CPO. At PN22, and persisting as long as 4 months, chronic developmental exposure to 0.18 mg/kg/d or 0.25 mg/kg/d CPO resulted in perinuclear vacuolization of cells in a discrete area of the neocortex and irregular distribution of neurons in the cortical plate, with an increase in the number of affected cells at 0.25 mg/kg/d. Third, we describe a transgenic mouse model in which human transgenes encoding either hPON1 Q 1 9 2 or hPON1 R 1 9 2 were expressed at equal levels in place of mouse PON1. The developmental onset ofexpression followed the mouse time course and was identical for the two transgenes, allowing these mice to be used to assess the importance of the Q192R polymorphism during development. Adult mice expressing hPON1 R 1 9 2 were significantly more resistant than hPON1 Q 1 9 2 mice to CPO toxicity. Our studies indicate that children less than 2 years old, especially those homozygous for PON1 Q 1 9 2 , would be predicted to be particularly susceptible to CPO toxicity.
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Paraoxonase 1 (PON1) attenuates macrophage oxidative status: studies in PON1 transfected cells and in PON1 transgenic mice.
Atherosclerosis, 2005Co-Authors: Orit Rozenberg, Diana M Shih, Michael AviramAbstract:Abstract Objective: High density lipoprotein (HDL)-associated paraoxonase 1 (PON1), hydrolyzes oxidized lipids in oxidized low density lipoprotein (LDL) and thus protects against atherosclerosis development. Increased susceptibility to atherosclerosis observed in PON1 knockout (PON1 0 ) mice was associated with increased LDL lipid peroxidation as well as increased macrophage oxidative stress. Thus, the aim of the present study is to characterize the direct effect of PON1 on oxidative status processes in macrophages. Methods and results: We used in vitro and in vivo models of PON1 expression in macrophages, as PON1 is not synthesized by these cells. Peritoneal macrophages (MPM) harvested from PON1 0 mice were transfected with human (hPON1). These cells exhibited reduced total peroxide levels by 47% and decreased capacity to release superoxide anions by 69%, associated with a small but significant increment of the reduced form of glutathione (GSH), a major cellular anti-oxidant, compared to control cells. MPM were also harvested from PON1 transgenic (PON1Tg) mice. Unexpectedly, these cells expressed hPON1 (mRNA and activity). Compared to MPM derived from control C57BL/6J mice, PON1Tg mouse MPM exhibited 35% decreased cellular total peroxide levels, decreased capacity to produce superoxide anions and 47% decreased capacity to oxidize LDL. PON1Tg mouse MPM were also characterized by 51% increased levels of GSH, compared to control MPM. Similarly, MPM harvested from PON1Tg on the genetic background of the atherosclerotic apolipoprotein E knockout (PON1Tg/E 0 ) mice also exhibited decreased oxidative stress, compared to E 0 mouse MPM. Aortas obtained from these mice were characterized by decreased lipid peroxide levels, decreased capacity to oxidize LDL, and also increased GSH levels, compared to aortas obtained from E 0 mice. The decreased macrophage and aortic oxidative stress in PON1Tg/E 0 mice was associated with 2.7-fold decreased atherosclerotic lesion size in comparison to E 0 mice. Conclusions: PON1 directly reduced macrophage and aortic oxidative status, which was associated with decreased superoxide anion production and increased glutathione content. These phenomena could be responsible for the observed attenuated atherosclerosis development in PON1Tg mice in comparison to control mice.
