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Abstract Background Paraoxonase-1 (PON1), a lactonase synthesized by the liver, circulates in blood bound to high-density lipoproteins (HDL). This enzyme is thought to degrade oxidized phospholipids and play an important role in the organism's antioxidant and anti-inflammatory system. Chronic liver diseases are characterized by decreased serum PON1 activity. The aim of the present study was to investigate the compositional changes in HDL that could influence PON1 activity in liver impairment. Methods The study was performed in samples from five patients with advanced liver cirrhosis and with preserved renal function, chosen on the basis of having low serum PON1 activity and high serum PON1 concentration. As a control group, we accessed five healthy volunteers from among our hospital staff. Lipid and protein compositional analysis of lipoprotein particles were done by high-performance liquid chromatography, gel electrophoresis, and Western-Blot. Results HDL particles from cirrhotic patients had an increased phospholipid content that was inversely correlated to PON1 activity. The HDL particles contained high levels of PON1 that corresponded, in part, to an immunoreactive protein of high molecular weight (55 kDa) not present in control subjects. This protein was identified as glycosylated PON1 and was also present in biopsies from patients with steatosis and from rats with CCl4-induced hepatic impairment. These changes were associated with an increased plasma concentration of markers of oxidative stress, inflammation and fibrogenesis. Conclusion Abnormalities in the composition of lipids and proteins of HDL particles, including PON1 glycosylation, are associated with the decrease in serum PON1 activity in patients with liver disease. These alterations may adversely affect the protective role of HDL against oxidative stress and inflammation in these patients.

Abstract Background Paraoxonase-1 (PON1) is an antioxidant enzyme that attenuates the production of the monocyte chemoattractant protein-1 (MCP-1) in vitro. Although oxidation and inflammation are closely related processes, the association between PON1 and MCP-1 has not been completely characterised due, probably, to that the current use of synthetic substrates for PON1 measurement limits the interpretation of the data. In the present study, we explored the relationships between the circulating levels of PON1 and MCP-1 in human immunodeficiency virus-infected patients in relation to the multifunctional capabilities of PON1. Methods We measured selected variables in 227 patients and in a control group of 409 participants. Serum PON1 esterase and lactonase activities were measured as the rates of hydrolysis of paraoxon and of 5-(thiobutyl)-butyrolactone, respectively. Oxidised LDL and MCP-1 concentrations were determined by enzyme-linked immunosorbent assay. High-density lipoproteins cholesterol, apolipoprotein A-I, and C-reactive protein concentrations were measured by standard automated methods. Results There were significant relationships between PON1 activity and several indices of oxidation and inflammation in control subjects and in infected patients. However, these relationships varied not only with disease status but also on the type of substrate used for PON1 measurement. Conclusion The present study is a cautionary tale highlighting that results of clinical studies on PON1 may vary depending on the methods used as well as the disease studied. Until more specific methods using physiologically-akin substrates are developed for PON1 measurement, we suggest the simultaneous employment of at least two different substrates in order to improve the reliability of the results obtained.

Abstract Background Paraoxonase-1 (PON1) is an antioxidant enzyme synthesized by the liver. It protects against liver impairment and attenuates the production of the pro-inflammatory monocyte chemoattractant protein-1 (MCP-1). We investigated the relationships between hepatic PON1 and MCP-1 expression in rats with liver disease and explored the possible molecular mechanisms involved. Methods CCl4 was administered for up to 12 weeks to induce liver damage. Serum and hepatic levels of PON1 and MCP-1, their gene and protein expression, nuclear transcription factors, and histological and biochemical markers of liver impairment were measured. Results High levels of PON1 and MCP-1 expression were observed at 12th week in the hepatocytes surrounding the fibrous septa and inflammatory areas. CCl4-administered rats had an increased hepatic PON1 concentration that was related to decreased gene transcription and inhibited protein degradation. Decreased PON1 gene transcription was associated with PPARδ expression. These changes were accompanied by increased hepatic MCP-1 concentration and gene expression. There were significant direct relationships between hepatic PON1 and MCP-1 concentrations (P = 0.005) and between PON1 and the amount of activated stellate cells (P = 0.001). Conclusion Our results from this experimental model suggest a hepato-protective role for PON1 against inflammation, fibrosis and liver disease mediated by MCP-1.

Background: Deployment-related neurotoxicant exposures are implicated in the etiology of Gulf War illness (GWI), the multisymptom condition associated with military service in the 1990-1991 Gulf War (GW). A Q/R polymorphism at position 192 of the paraoxonase (PON)-1 enzyme produce PON1 192 variants with different capacities for neutralizing specific chemicals, including certain acetylcholinesterase inhibitors., Methods: We evaluated PON1 192 status and GW exposures in 295 GWI cases and 103 GW veteran controls. Multivariable logistic regression determined independent associations of GWI with GW exposures overall and in PON1 192 subgroups. Exact logistic regression explored effects of exposure combinations in PON1 192 subgroups., Results: Hearing chemical alarms (proxy for possible nerve agent exposure) was associated with GWI only among RR status veterans (OR = 8.60, p = 0.014). Deployment-related skin pesticide use was associated with GWI only among QQ (OR = 3.30, p = 0.010) and QR (OR = 4.22, p < 0.001) status veterans. Exploratory assessments indicated that chemical alarms were associated with GWI in the subgroup of RR status veterans who took pyridostigmine bromide (PB) (exact OR = 19.02, p = 0.009) but not RR veterans who did not take PB (exact OR = 0.97, p = 1.00). Similarly, skin pesticide use was associated with GWI among QQ status veterans who took PB (exact OR = 6.34, p = 0.001) but not QQ veterans who did not take PB (exact OR = 0.59, p = 0.782)., Conclusion: Study results suggest a complex pattern of PON1 192 exposures and exposure-exposure interactions in the development of GWI.

The kinetic analysis of esterase inhibition by acylating compounds (organophosphorus, carbamates and sulfonylfluorides) sometimes cannot yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In this work kinetic data were obtained for demeton-S-methyl (DSM) with human acetylcholinesterase in two kinds of experiments: (a) time progressive inhibition with a range of concentrations, (b) progressive spontaneous reactivation starting with pre-inhibited enzyme. DSM is an organophosphorus compound used as pesticide and considered a model for studying the dermal exposure of nerve agents such as VX gas. A kinetic model equation was deduced with four different molecular phenomena occurring simultaneously: (1) inhibition; (2) spontaneous reactivation; (3) aging; and (4) ongoing inhibition (inhibition during the substrate reaction). A 3D fit of the model was applied to analyze the inhibition experimental data. The best-fitting model is compatible with a sensitive enzymatic entity. The second-order rate constant of inhibition (ki = 0.0422 μM -1  min -1 ), the spontaneous reactivation constant (ks = 0.0202 min -1 ) and the aging constant (kg = 0.0043 min -1 ) were simultaneously estimated. As an example for testing the model and approach, it was tested also in the presence of 5 % ethanol (conditions as previously used in the literature), the best fitting model is compatible with two apparent sensitive enzymatic entities (17 % and 83 %) and only one spontaneously reactivates and ages. The corresponding second-order rate constants of inhibition (ki = 0.0354 and 0.0119 μM -1  min -1 ) and the spontaneous reactivation and aging constants for the less sensitive component (kr = 0.0203 min -1 and kg = 0.0088 min -1 ) were estimated. The results were also consistent with a significant ongoing inhibition. These parameters were similar to those deduced in spontaneous reactivation experiments of the pre-inhibited samples with DSM in the absence or presence of ethanol. The two apparent components fit was interpreted by an equilibrium between ethanol-free and ethanol-bound enzyme. The consistency of results in inhibition and in spontaneous reactivation experiments was considered an internal validation of the methodology and the conclusions., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jorge Estevez reports equipment, drugs, or supplies and travel were provided by Government of Spain Ministry of Education and Vocational Training., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Convergent adaptation to the same environment by multiple lineages frequently involves rapid evolutionary change at the same genes, implicating these genes as important for environmental adaptation. Such adaptive molecular changes may yield either change or loss of protein function; loss of function can eliminate newly deleterious proteins or reduce energy necessary for protein production. We previously found a striking case of recurrent pseudogenization of the Paraoxonase 1 (Pon1) gene among aquatic mammal lineages-Pon1 became a pseudogene with genetic lesions, such as stop codons and frameshifts, at least four times independently in aquatic and semiaquatic mammals. Here, we assess the landscape and pace of pseudogenization by studying Pon1 sequences, expression levels, and enzymatic activity across four aquatic and semiaquatic mammal lineages: pinnipeds, cetaceans, otters, and beavers. We observe in beavers and pinnipeds an unexpected reduction in expression of Pon3, a paralog with similar expression patterns but different substrate preferences. Ultimately, in all lineages with aquatic/semiaquatic members, we find that preceding any coding-level pseudogenization events in Pon1, there is a drastic decrease in expression, followed by relaxed selection, thus allowing accumulation of disrupting mutations. The recurrent loss of Pon1 function in aquatic/semiaquatic lineages is consistent with a benefit to Pon1 functional loss in aquatic environments. Accordingly, we examine diving and dietary traits across pinniped species as potential driving forces of Pon1 functional loss. We find that loss is best associated with diving activity and likely results from changes in selective pressures associated with hypoxia and hypoxia-induced inflammation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

A wealth of evidence suggests that Lipoprotein-associated phospholipase A2 (Lp-PLA2) plays a relevant role in atherogenesis and inflammation, which in turn are associated with the risk of developing dementia. The aim of this study was to evaluate whether serum Lp-PLA2 activity might be an early and/or late biomarker for different forms of dementia. Serum Lp-PLA2 activity was assessed in older patients with mild cognitive impairment (MCI, n = 166; median clinical follow-up = 29 months), Late-Onset Alzheimer's disease (LOAD, n = 176), vascular dementia (VAD, n = 43), dementia characterized by an overlap between LOAD and VAD (AD-VAD MIXED dementia) ( n = 136), other dementia subtypes ( n = 45), and cognitively normal controls ( n = 151). We found a significant trend towards higher levels of Lp-PLA2 activity in VAD compared with the other groups (ANOVA, p = 0.028). Similarly, Lp-PLA2 activity was greater in MCI converting to VAD compared with those that did not or did convert to the other types of dementia (ANOVA, p = 0.011). After adjusting for potential confounders, high levels of Lp-PLA2 activity were associated with the diagnosis of VAD (O.R. = 2.38, 95% C.I. = 1.06-5.10), but not with other types of dementia. Our data suggest that increased serum Lp-PLA2 activity may represent a potential biomarker for the diagnosis of VAD.

Background: Paraoxonase 2 (PON2) is an intracellular antioxidant enzyme located at the inner mitochondrial membrane. Previous studies have found PON2 to be an important antioxidant in a variety of cellular systems, such as the cardiovascular and renal system. Recent work has also suggested that PON2 plays an important role in the central nervous system (CNS), as decreased PON2 expression in the CNS leads to higher oxidative stress and subsequent cell toxicity. However, the precise role of PON2 in the CNS is still largely unknown, and what role it may play in specific regions of the brain remains unexamined. Dopamine metabolism generates considerable oxidative stress and antioxidant function is critical to the survival of dopaminergic neurons, providing a potential mechanism for PON2 in the dopaminergic system., Methods: In this study, we investigated the role of PON2 in the dopaminergic system of the mouse brain by comparing transcript and protein expression of dopaminergic-related genes in wildtype (WT) and PON2 deficient (PON2-def) mouse striatum, and exposing WT cultured primary neurons to dopamine receptor agonists., Results: We found alterations in multiple key dopaminergic genes at the transcript level, however many of these changes were not observed at the protein level. In cultured neurons, PON2 mRNA and protein were increased upon exposure to quinpirole, a dopamine receptor 2/3 (DRD2/3) agonist, but not fenoldopam, a dopamine receptor 1/5 (DRD1/5) agonist, suggesting a receptor-specific role in dopamine signaling., Conclusions: Our findings suggest PON2 deficiency significantly impacts the dopaminergic system at the transcript level and may play a role in mitigating oxidative stress in this system further downstream through dopamine receptor signaling., (© 2022. The Author(s).)

Aim: To review and compare the PON-1 arylesterase activity between coronary artery disease (CAD) and non-CAD patients., Methods: Data were obtained by searching MEDLINE and Scopus for all investigations published between January 1, 2000 and March 1, 2021 comparing PON-1 arylesterase activity between CAD and controls., Results: Twenty studies, based on 5417 patients, met the inclusion criteria and were included in the analysis. A random effect model revealed that PON-1 arylesterase activity was significantly lower in the CAD group compared to controls (SMD = -0.587, 95%CI = -0.776 to -0.339, p < 0.0001, I 2 = 92.3%). In CAD patients, the PON-1 arylesterase activity was significantly higher among CAD patients without diabetes mellitus (DM) compared to those with diabetes (SMD: 0.235, 95% CI: 0.014 to 0.456, p = 0.03, I 2 = 0%)., Conclusions: PON-1 activity is significantly lower in CAD patients, and those without DM presented a significantly higher PON-1 arylesterase activity., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2022 Marco Zuin et al.)

Paraoxonase 2 (PON2) is an intracellular antioxidant enzyme shown to play an important role in mitigating oxidative stress in the brain. Oxidative stress is a common mechanism of toxicity for neurotoxicants and is increasingly implicated in the etiology of multiple neurological diseases. While PON2 deficiency increases oxidative stress in the brain in-vitro, little is known about its effects on behavior in-vivo and what global transcript changes occur from PON2 deficiency. We sought to characterize the effects of PON2 deficiency on behavior in mice, with an emphasis on locomotion, and evaluate transcriptional changes with RNA-Seq. Behavioral endpoints included home-cage behavior (Noldus PhenoTyper), motor coordination (Rotarod) and various gait metrics (Noldus CatWalk). Home-cage behavior analysis showed PON2 deficient mice had increased activity at night compared to wildtype controls and spent more time in the center of the cage, displaying a possible anxiolytic phenotype. PON2 deficient mice had significantly shorter latency to fall when tested on the rotarod, suggesting impaired motor coordination. Minimal gait alterations were observed, with decreased girdle support posture noted as the only significant change in gait with PON2 deficiency. Beyond one home-cage metric, no significant sex-based behavioral differences were found in this study. Finally, A subset of samples were utilized for RNA-Seq analysis, looking at three discrete brain regions: cerebral cortex, striatum, and cerebellum. Highly regional- and sex-specific changes in RNA expression were found when comparing PON2 deficient and wildtype mice, suggesting PON2 may play distinct regional roles in the brain in a sex-specific manner. Taken together, these findings demonstrates that PON2 deficiency significantly alters the brain on both a biochemical and phenotypic level, with a specific impact on motor function. These data have implications for future gene-environment toxicological studies and warrants further investigation of the role of PON2 in the brain., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Motor deficits can significantly affect the completion of daily life activities and have a negative impact on quality of life. Consequently, motor function is an important behavioral endpoint to measure for in vivo pathophysiologic studies in a variety of research areas, such as toxicant exposure, drug development, disease characterization, and transgenic phenotyping. Evaluation of motor function is also critical to the interpretation of cognitive behavioral assays, as many rely on intact motor abilities to derive meaningful data. As such, gait analysis is an important component of behavioral research and can be achieved by manual or video-assisted methods. Manual gait analysis methods, however, are prone to observer bias and are unable to capture many critical parameters. In contrast, automated video-assisted gait analysis can quickly and reliably assess gait and locomotor abnormalities that were previously difficult to collect manually. Here, we describe the evaluation of gait and locomotion in rodents using the automated Noldus CatWalk XT system. We include a step-by-step guide for running an experiment using the CatWalk XT system and discuss theory and considerations when evaluating rodent gait. The protocol and discussion provided here act as a supplemental resource to the manual for this commercially available system and can assist CatWalk users in their experimental design and implementation. © 2021 Wiley Periodicals LLC., (© 2021 Wiley Periodicals LLC.)

Rates of preterm birth and low birthweight continue to rise in the United States and pose a significant public health problem. Although a variety of environmental exposures are known to contribute to these and other adverse birth outcomes, there has been a limited success in developing policies to prevent these outcomes. A better characterization of the complexities between multiple exposures and their biological responses can provide the evidence needed to inform public health policy and strengthen preventative population-level interventions. In order to achieve this, we encourage the establishment of an interdisciplinary data science framework that integrates epidemiology, toxicology and bioinformatics with biomarker-based research to better define how population-level exposures contribute to these adverse birth outcomes. The proposed interdisciplinary research framework would 1) facilitate data-driven analyses using existing data from health registries and environmental monitoring programs; 2) develop novel algorithms with the ability to predict which exposures are driving, in this case, adverse birth outcomes in the context of simultaneous exposures; and 3) refine biomarker-based research, ultimately leading to new policies and interventions to reduce the incidence of adverse birth outcomes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Brain oxidative lipid damage and inflammation are common in neurodegenerative diseases such as Alzheimer's disease (AD). Paraoxonase-1 and -3 (PON1 and PON3) protein expression was demonstrated in tissue with no PON1 or PON3 gene expression. In the present study, we examine differences in PON1 and PON3 protein expression in the brain of a mouse model of AD., Methods: we used peroxidase- and fluorescence-based immunohistochemistry in five brain regions (olfactory bulb, forebrain, posterior midbrain, hindbrain and cerebellum) of transgenic (Tg2576) mice with the Swedish mutation (KM670/671NL) responsible for a familial form of AD and corresponding wild-type mice., Results: We found intense PON1 and PON3-positive staining in star-shaped cells surrounding Aβ plaques in all the studied Tg2576 mouse-brain regions. Although we could not colocalize PON1 and PON3 with astrocytes (star-shaped cells in the brain), we found some PON3 colocalization with microglia., Conclusions: These results suggest that (1) PON1 and PON3 cross the blood-brain barrier in discoidal high-density lipoproteins (HDLs) and are transferred to specific brain-cell types; and (2) PON1 and PON3 play an important role in preventing oxidative stress and lipid peroxidation in particular brain-cell types (likely to be glial cells) in AD pathology and potentially in other neurodegenerative diseases as well.

The protein composition of high-density lipoprotein (HDL) is extremely fluid. The quantity and quality of protein constituents drive the multiple biological functions of these lipoproteins, which include the ability to contrast atherogenesis, sustained inflammation, and toxic effects of reactive species. Several diseases where inflammation and oxidative stress participate in the pathogenetic process are characterized by perturbation in the HDL proteome. This change inevitably affects the functionality of the lipoprotein. An enlightening example in this frame comes from the literature on Alzheimer's disease (AD). Growing lines of epidemiological evidence suggest that loss of HDL-associated proteins, such as lipoprotein phospholipase A2 (Lp-PLA2), glutathione peroxidase-3 (GPx-3), and paraoxonase-1 and paraoxonase-3 (PON1, PON3), may be a feature of AD, even at the early stage. Moreover, the decrease in these enzymes with antioxidant/defensive action appears to be accompanied by a parallel increase of prooxidant and proinflammatory mediators, in particular myeloperoxidase (MPO) and serum amyloid A (SAA). This type of derangement of balance between two opposite forces makes HDL dysfunctional, i.e., unable to exert its "natural" vasculoprotective property. In this review, we summarized and critically analyzed the most significant findings linking HDL accessory proteins and AD. We also discuss the most convincing hypothesis explaining the mechanism by which an observed systemic occurrence may have repercussions in the brain., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Francesca Zimetti et al.)

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 PON1 192 genotype and plasma PON1 activity levels. While our previous studies with PON1 status demonstrated that both PON1 192 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., (© 2021 Wiley Periodicals LLC.)

Several lines of epidemiological evidence link increased levels of high-density lipoprotein-cholesterol (HDL-C) with lower risk of Alzheimer's disease (AD). This observed relationship might reflect the beneficial effects of HDL on the cardiovascular system, likely due to the implication of vascular dysregulation in AD development. The atheroprotective properties of this lipoprotein are mostly due to its proteome. In particular, apolipoprotein (Apo) A-I, E, and J and the antioxidant accessory protein paraoxonase 1 (PON1), are the main determinants of the biological function of HDL. Intriguingly, these HDL constituent proteins are also present in the brain, either from in situ expression, or derived from the periphery. Growing preclinical evidence suggests that these HDL proteins may prevent the aberrant changes in the brain that characterize AD pathogenesis. In the present review, we summarize and critically examine the current state of knowledge on the role of these atheroprotective HDL-associated proteins in AD pathogenesis and physiopathology.

At present, the etiopathogenesis of Alzheimer's disease (AD), the most common form of dementia, remains far to be fully deciphered. In the recent years, also the centrality of amyloid-β peptide in the pathogenesis of the neurodegenerative disease has been questioned and other hypotheses have been advanced. Notably, a common denominator of many of these theoretical models is represented by oxidative stress, which is widely proposed to play a role in the disease initiation and/or progression. Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme that endows its carrier with multiple biological functions, including the ability to contrast oxidative damage to lipid components of lipoproteins and cells and protect from toxicity of specific organophosphorus pesticides. The peculiar multi-functionality nature of PON1 might be the key for explaining the vast epidemiological data showing a close association between low serum PON1 activity and risk of several diseases, including cardiovascular and neurodegenerative diseases, in particular AD. In this review, we discuss the possible link between PON1 with AD pathogenesis and we hypothesize eventual mechanistic pathways that could account from epidemiological observations. We also highlight the methodological issue limitation in PON1 studies that still impede to give a definitive and certain picture of its effective biological impact on human health including AD.

Mammals diversified by colonizing drastically different environments, with each transition yielding numerous molecular changes, including losses of protein function. Though not initially deleterious, these losses could subsequently carry deleterious pleiotropic consequences. We have used phylogenetic methods to identify convergent functional losses across independent marine mammal lineages. In one extreme case, Paraoxonase 1 ( PON1 ) accrued lesions in all marine lineages, while remaining intact in all terrestrial mammals. These lesions coincide with PON1 enzymatic activity loss in marine species' blood plasma. This convergent loss is likely explained by parallel shifts in marine ancestors' lipid metabolism and/or bloodstream oxidative environment affecting PON1's role in fatty acid oxidation. PON1 loss also eliminates marine mammals' main defense against neurotoxicity from specific man-made organophosphorus compounds, implying potential risks in modern environments., (Copyright © 2018, American Association for the Advancement of Science.)

The paraoxonases (PONs) are a three-gene family which includes PON1, PON2, and PON3. PON1 and PON3 are synthesized primarily in the liver and a portion is secreted in the plasma, where they are associated with high-density lipoproteins (HDLs), while PON2 is an intracellular enzyme, expressed in most tissues and organs, including the brain. PON1 received its name from its ability to hydrolyze paraoxon, the active metabolite of the organophosphorus (OP) insecticide parathion, and also more efficiently hydrolyzes the active metabolites of several other OPs. PON2 and PON3 do not have OP-esterase activity, but all PONs are lactonases and are capable of hydrolyzing a variety of lactones, including certain drugs, endogenous compounds, and quorum-sensing signals of pathogenic bacteria. In addition, all PONs exert potent antioxidant effects. PONs play important roles in cardiovascular diseases and other oxidative stress-related diseases, modulate susceptibility to infection, and may provide neuroprotection (PON2). Hence, significant attention has been devoted to their modulation by a variety of dietary, pharmacological, lifestyle, or environmental factors. A number of metals have been shown in in vitro, animal, and human studies to mostly negatively modulate expression of PONs, particularly PON1, the most studied in this regard. In addition, different levels of expression of PONs may affect susceptibility to toxicity and neurotoxicity of metals due to their aforementioned antioxidant properties.

Paraoxonase 2 (PON2) is a member of the paraoxonase gene family also comprising PON1 and PON3. PON2 functions as a lactonase and exhibits anti-bacterial as well as antioxidant properties. At the cellular level, PON2 localizes to the mitochondrial and endoplasmic reticulum membranes where it scavenges reactive oxygen species. PON2 is of particular interest as it is the only paraoxonase expressed in brain tissue and appears to play a critical role in mitigating oxidative stress in the brain. The aim of this study was to investigate the expression of PON2 at the protein and mRNA level in the brain and liver of mice through development to identify potential age windows of susceptibility to oxidative stress, as well as to compare expression of hepatic PON2 to expression of PON1 and PON3. Overall, PON2 expression in the brain was lower in neonatal mice and increased with age up to postnatal day (PND) 21, with a significant decrease observed at PND 30 and 60. In contrast, the liver showed continuously increasing levels of PON2 with age, similar to the patterns of PON1 and PON3. PON2 protein levels were also investigated in brain samples from non-human primates, with PON2 increasing with age up to the infant stage and decreasing at the juvenile stage, mirroring the results observed in the mouse brain. These variable expression levels of PON2 suggest that neonatal and young adult animals may be more susceptible to neurological insult by oxidants due to lower levels of PON2 in the brain., Competing Interests: STATEMENT The authors declare no conflicts of interest., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Paraoxonase-1 (PON1), an esterase/lactonase primarily associated with plasma high-density lipoprotein (HDL), was the first member of this family of enzymes to be characterized. Its name was derived from its ability to hydrolyze paraoxon, the toxic metabolite of the insecticide parathion. Related enzymes PON2 and PON3 were named from their evolutionary relationship with PON1. Mice with each PON gene knocked out were generated at UCLA and have been key for elucidating their roles in organophosphorus (OP) metabolism, cardiovascular disease, innate immunity, obesity, and cancer. PON1 status, determined with two-substrate analyses, reveals an individual's functional Q192R genotype and activity levels. The three-dimensional structure for a chimeric PON1 has been useful for understanding the structural properties of PON1 and for engineering PON1 as a catalytic scavenger of OP compounds. All three PONs hydrolyze microbial N-acyl homoserine lactone quorum sensing factors, quenching Pseudomonas aeruginosa's pathogenesis. All three PONs modulate oxidative stress and inflammation. PON2 is localized in the mitochondria and endoplasmic reticulum. PON2 has potent antioxidant properties and is found at 3- to 4-fold higher levels in females than males, providing increased protection against oxidative stress, as observed in primary cultures of neurons and astrocytes from female mice compared with male mice. The higher levels of PON2 in females may explain the lower frequency of neurological and cardiovascular diseases in females and the ability to identify males but not females with Parkinson's disease using a special PON1 status assay. Less is known about PON3; however, recent experiments with PON3 knockout mice show them to be susceptible to obesity, gallstone formation and atherosclerosis. Like PONs 1 and 2, PON3 also appears to modulate oxidative stress. It is localized in the endoplasmic reticulum, mitochondria and on HDL. Both PON2 and PON3 are upregulated in cancer, favoring tumor progression through mitochondrial protection against oxidative stress and apoptosis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Acute and chronic exposures to widely used organophosphorus (OP) insecticides are common. Children's detoxification mechanisms are not well developed until several years after birth. The increased cases of neurodevelopmental disorders in children, together with their increased susceptibility to OP neurotoxicity cannot be explained by genetic factors alone but could be related to gene-environment interactions. Paraoxonase-1 (PON1) is an enzyme that can detoxify OPs but its catalytic efficiency for hydrolysis to certain OPs is modulated by the Q192R polymorphism. Studies with animals have provided important information on the role of PON1 in protecting against gestational and postnatal toxicity to OPs. The PON1Q192 allele is less efficient in hydrolyzing certain OPs than the PON1R192 allele. Maternal PON1 status (PON1 activity levels, the most important measurement, and functional Q192R phenotype) modulates the detrimental effects of exposure to the OP chlorpyrifos oxon on fetal brain gene expression and biomarkers of exposure. Epidemiologic studies suggest that children from mothers with lower PON1 status who were in contact with OPs during pregnancy tend to show smaller head circumference at birth and adverse effects in cognitive function during childhood. Infants and children are vulnerable to OP toxicity. The detrimental consequences of OPs on neurodevelopment can lead to future generations with permanent cognitive problems and susceptibility to develop neurodegenerative diseases. Improved methods using mass spectrometry to monitor OP-adducted biomarker proteins are needed and will be extremely helpful in early life biomonitoring, while measurement of PON1 status as a biomarker of susceptibility will help identify mothers and children highly sensitive to OPs. The use of adductomics instead of enzymatic activity assays for biomonitoring OP exposures have proved to provide several advantages, including the use of dried blood spots, which would facilitate monitoring newborn babies and children., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Patients with autoimmune diseases have a significantly increased risk of developing cardiovascular disease. In disease, high-density lipoprotein (HDL) particles lose their anti-inflammatory and antioxidant properties and become dysfunctional. The purpose of this study was to test the hypothesis that alterations in the HDL proteomic profile are associated with subclinical atherosclerosis and HDL dysfunction in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and type 1 diabetes. Targeted proteomics was used to quantify the relative abundance of 18 proteins in HDL from SLE patients with and without atherosclerotic plaque detectable by carotid ultrasound. Changes in the proteomic profile were compared against the in vitro ability of HDL to protect against lipid oxidation. The same proteins were quantified in HDL from patients with type 1 diabetes with or without coronary artery calcification as determined by computed tomography. In each population, paraoxonase-3 (PON3), a potent antioxidant protein, was depleted from the HDL of patients with subclinical atherosclerosis. PON3 expression in HDL was positively correlated with HDL antioxidant function. These results suggest that PON3 may be an important protein in preventing atherosclerosis and highlight the importance of antioxidant proteins in the prevention of atherosclerosis in vivo.

Homocysteinylation of lysine residues by homocysteine thiolactone (HCTL), a reactive homocysteine metabolite, results in protein aggregation and malfunction, and is a well-known risk factor for cardiovascular, autoimmune and neurological diseases. Human plasma paraoxonase-1 (PON1) and bleomycin hydrolase (Blmh) have been reported as the physiological HCTL detoxifying enzymes. However, the catalytic efficiency of HCTL hydrolysis by Blmh is low and not saturated at 20 mM HCTL. The catalytic efficiency of PON1 for HCTL hydrolysis is 100-fold lower than that of Blmh. A homocysteine thiolactonase (HCTLase) was purified from human liver and identified by mass spectrometry (MS) as the previously described human biphenyl hydrolase-like protein (BPHL). To further characterize this newly described HCTLase activity, BPHL was expressed in Escherichia coli and purified. The sequence of the recombinant BPHL (rBPHL) and hydrolytic products of the substrates HCTL and valacyclovir were verified by MS. We found that the catalytic efficiency (kcat/Km) of rBPHL for HCTL hydrolysis was 7.7 × 10(4) M(-1)s(-1), orders of magnitude higher than that of PON1 or Blmh, indicating a more significant physiological role for BPHL in detoxifying HCTL.

HDL-associated paraoxonase-1 (PON1) is an enzyme whose activity is associated with cerebrovascular disease. Common PON1 genetic variants have not been consistently associated with cerebrovascular disease. Rare coding variation that likely alters PON1 enzyme function may be more strongly associated with stroke. The National Heart, Lung, and Blood Institute Exome Sequencing Project sequenced the coding regions (exomes) of the genome for heart, lung, and blood-related phenotypes (including ischemic stroke). In this sample of 4,204 unrelated participants, 496 had verified, noncardioembolic ischemic stroke. After filtering, 28 nonsynonymous PON1 variants were identified. Analysis with the sequence kernel association test, adjusted for covariates, identified significant associations between PON1 variants and ischemic stroke (P = 3.01 × 10(-3)). Stratified analyses demonstrated a stronger association of PON1 variants with ischemic stroke in African ancestry (AA) participants (P = 5.03 × 10(-3)). Ethnic differences in the association between PON1 variants with stroke could be due to the effects of PON1Val109Ile (overall P = 7.88 × 10(-3); AA P = 6.52 × 10(-4)), found at higher frequency in AA participants (1.16% vs. 0.02%) and whose protein is less stable than the common allele. In summary, rare genetic variation in PON1 was associated with ischemic stroke, with stronger associations identified in those of AA. Increased focus on PON1 enzyme function and its role in cerebrovascular disease is warranted.

Objectives: To investigate the relationships between serum paraoxonase-1 (PON1), insulin resistance, and metabolic syndrome (MetS) in childhood obesity., Design and Methods: We studied 110 obese children and 36 non-obese children with a similar gender and age distribution. We measured serum PON1 activity against 5-thiobutyl butyrolactone (TBBLase) and against paraoxon (paraoxonase). PON1 concentration was measured separately as were the levels of several standard metabolic variables. The homeostasis model assessment (HOMA) index was calculated as an estimate of insulin resistance., Results: TBBLase was significantly decreased in obese children (P=0.008), while paraoxonase activity and PON1 concentrations showed non-significant trends towards decrease and increase, respectively (P=0.054 and P=0.060). TBBLase and paraoxonase specific activities were significantly decreased (P=0.004 and P=0.018, respectively). TBBLase specific activity was inversely associated with BMI, percentage body fat, insulin, HOMA, triglycerides, and C-reactive protein, and directly associated with HDL-cholesterol. Paraoxonase specific activity showed similar associations with BMI, percentage fat, HDL-cholesterol, and C-reactive protein. Obese children with MetS had lower TBBLase activities than obese children without MetS (P=0.018). Linear regression analyses showed that TBBLase was independently associated with HDL-cholesterol, BMI, percentage body fat and PON155 polymorphism, but paraoxonase activity was associated only with PON1192 polymorphism., Conclusions: Our results suggest that PON1 may play a role in the onset and development of metabolic alterations in childhood obesity leading to diabetes and cardiovascular disease later in life. However, being derived from statistical association study, this finding cannot be seen as showing cause-effect., (© 2013.)

PON1 is a key component of high-density lipoproteins (HDLs) and is at least partially responsible for HDL's antioxidant/atheroprotective properties. PON1 is also associated with numerous human diseases, including cardiovascular disease, Parkinson's disease and cancer. In addition, PON1 metabolizes a broad variety of substrates, including toxic organophosphorous compounds, statin adducts, glucocorticoids, the likely atherogenic L-homocysteine thiolactone and the quorum-sensing factor of Pseudomonas aeruginosa. Numerous cardiovascular and antidiabetic pharmacologic agents, dietary macronutrients, lifestyle factors and antioxidant supplements affect PON1 expression and enzyme activity levels. Owing to the importance of PON1 to HDL function and its individual association with diverse human diseases, pharmacogenomic interactions between PON1 and the various factors that alter its expression and activity may represent an important therapeutic target for future investigation.

Purpose: To investigate the relationships between the levels of the antioxidant enzyme paraoxonase-1 (PON1) and corneal endothelial alterations in patients with chronic obstructive pulmonary disease (COPD) undergoing cataract surgery., Methods: We studied 172 patients with cataract attending our ophthalmology clinic. Based on spirometric analysis, they were segregated into two groups, 110 (64%) with COPD and 62 (36%) without COPD. Corneal endothelial cell morphology was examined by widefield noncontact specular microscopy, which allows measurements of endothelial cell density (ECD), hexagonality, and endothelial cell size coefficient of variation (ECCV). Corneal thickness was measured by noncontact pachimetry. PON1 and plasma TNFα concentrations were analyzed by ELISA. Serum PON1 activity was analyzed by spectrophotometry., Results: Patients with COPD had significant decreases in ECD, hexagonality, and corneal thickness, and a significant increase in ECCV. They also had significant decreases in serum PON1 activity but not in PON1 concentration. Serum PON1 activity showed a significant direct association with ECD, and an inverse association with corneal thickness., Conclusions: Results of the present study suggest that PON1 may be involved in the pathophysiology of corneal endothelial alterations in patients with COPD.

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., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)