Reduction of Paraoxonase Expression Followed by Inactivation across Independent Semiaquatic Mammals Suggests Stepwise Path to Pseudogenization.

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.

Extended interaction networks with HCV protease NS3-4A substrates explain the lack of adaptive capability against protease inhibitors.

Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168 To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.

Inhibition with simultaneous spontaneous reactivation and aging of acetylcholinesterase by organophosphorus compounds: Demeton-S-methyl as a model.

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.

Dietary cholesterol increases paraoxonase 1 enzyme activity.

HDL-associated paraoxonase 1 (PON1) activity has been consistently associated with cardiovascular and other diseases. Vitamins C and E intake have previously been positively associated with PON1 in a subset of the Carotid Lesion Epidemiology and Risk (CLEAR) cohort. The goal of this study was to replicate these findings and determine whether other nutrient intake affected PON1 activity. To predict nutrient and mineral intake values, 1,402 subjects completed a standardized food frequency survey of their dietary habits over the past year. Stepwise regression was used to evaluate dietary and covariate effects on PON1 arylesterase activity. Five dietary components, cholesterol (P < 2.0 × 10(-16)), alcohol (P = 8.51 × 10(-8)), vitamin C (P = 7.97 × 10(-5)), iron (P = 0.0026), and folic acid (0.037) were independently predictive of PON1 activity. Dietary cholesterol was positively associated and predicted 5.5% of PON1 activity, second in variance explained. This study presents a novel finding of dietary cholesterol, iron, and folic acid predicting PON1 activity in humans and confirms prior reported associations, including that with vitamin C. Identifying and understanding environmental factors that affect PON1 activity is necessary to understand its role and that of HDL in human disease.

Regulatory Role of Phospholipids in Hepatitis C Virus Replication and Protein Function.

Positive-strand RNA viruses such as hepatitis C virus (HCV) hijack key factors of lipid metabolism of infected cells and extensively modify intracellular membranes to support the viral lifecycle. While lipid metabolism plays key roles in viral particle assembly and maturation, viral RNA synthesis is closely linked to the remodeling of intracellular membranes. The formation of viral replication factories requires a number of interactions between virus proteins and host factors including lipids. The structure-function relationship of those proteins is influenced by their lipid environments and lipids that selectively modulate protein function. Here, we review our current understanding on the roles of phospholipids in HCV replication and of lipid-protein interactions in the structure-function relationship of the NS5A protein. NS5A is a key factor in membrane remodeling in HCV-infected cells and is known to recruit phosphatidylinositol 4-kinase III alpha to generate phosphatidylinositol 4-phosphate at the sites of replication. The dynamic interplay between lipids and viral proteins within intracellular membranes is likely key towards understanding basic mechanisms in the pathobiology of virus diseases, the mode of action of specific antiviral agents and related drug resistance mechanisms.

Hyperosmotic Stress Allosterically Reconfigures Betaine Binding Pocket in BetP.

The transporter BetP in C. glutamicum is essential in maintaining bacterial cell viability during hyperosmotic stress and functions by co-transporting betaine and Na+ into bacterial cells. Hyperosmotic stress leads to increased intracellular K+ concentrations which in turn promotes betaine binding. While structural details of multiple end state conformations of BetP have provided high resolution snapshots, how K+ sensing by the C-terminal domain is allosterically relayed to the betaine binding site is not well understood. In this study, we describe conformational dynamics in solution of BetP using amide hydrogen/deuterium exchange mass spectrometry. These reveal how K+ alters conformation of the disordered C- and N-terminal domains to allosterically reconfigure transmembrane helices 3, 8, and 10 to enhance betaine interactions. A map of the betaine binding site, at near single amino acid resolution, reveals a critical extrahelical H-bond mediated by TM3 with betaine.

Engineered recombinant human paraoxonase 1 (rHuPON1) purified from Escherichia coli protects against organophosphate poisoning.

The high-density lipoprotein-associated enzyme paraoxonase 1 (PON1) hydrolyzes lactones, aromatic esters, and neurotoxic organophosphorus (OP) compounds, including insecticide metabolites and nerve agents. Experiments with mice lacking PON1 (PON1(-/-) mice) have established that plasma PON1 protects against chlorpyrifos/chlorpyrifos-oxon and diazinon/diazoxon (DZO) exposure but does not protect against parathion/paraoxon or nerve agents. The catalytic efficiency of PON1 determines whether or not it will protect against a given OP exposure. Expression of active recombinant human PON1 (rHuPON1) in Escherichia coli provides a system in which PON1 can be engineered to achieve a catalytic efficiency sufficient to protect against or treat specific OP exposures. Here, we describe the generation of highly purified engineered rHuPON1(K192) that protects against DZO exposure when injected into PON1(-/-) mice. The injected rHuPON1 is nontoxic, persists in serum for at least 2 days after injection, and provides protection against DZO exposures of at least three times the median lethal dose value.

Paraoxonase-1 (PON1) Status Analysis Using Non-Organophosphate Substrates.

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.

A Digital Twin Approach for Contextual Assistance for Surgeons During Surgical Robotics Training.

Minimally invasive robotic surgery copes with some disadvantages for the surgeon of minimally invasive surgery while preserving the advantages for the patient. Most commercially available robotic systems are telemanipulated with haptic input devices. The exploitation of the haptics channel, e.g., by means of Virtual Fixtures, would allow for an individualized enhancement of surgical performance with contextual assistance. However, it remains an open field of research as it is non-trivial to estimate the task context itself during a surgery. In contrast, surgical training allows to abstract away from a real operation and thus makes it possible to model the task accurately. The presented approach exploits this fact to parameterize Virtual Fixtures during surgical training, proposing a Shared Control Parametrization Engine that retrieves procedural context information from a Digital Twin. This approach accelerates a proficient use of the robotic system for novice surgeons by augmenting the surgeon's performance through haptic assistance. With this our aim is to reduce the required skill level and cognitive load of a surgeon performing minimally invasive robotic surgery. A pilot study is performed on the DLR MiroSurge system to evaluate the presented approach. The participants are tasked with two benchmark scenarios of surgical training. The execution of the benchmark scenarios requires basic skills as pick, place and path following. The evaluation of the pilot study shows the promising trend that novel users profit from the haptic augmentation during training of certain tasks.

Paraoxonase 1 status as a risk factor for disease or exposure.

Human paraoxonase 1 (PON1) has broad substrate specificity and has been shown to protect against exposure to some organophosphorus (OP) insecticides due to its ability to hydrolyze toxic metabolites of some organophosphorothioate insecticides. PON1 status has been shown to be important in protecting against vascular disease, presumably due to the not-as-yet fully characterized role of the three PON proteins in modulating oxidative stress. More recently, all three PONs (1, 2, and 3) have been shown to inactivate the quorum sensing factor N-(3-oxododecanoyl)-L: -homoserine lactone (3OC12-HSL) of Pseudomonas. Expression of human PON1 in Drosophila demonstrated the importance of PON1 in resistance to Pseudomonas infection. Many studies have examined only DNA single nucleotide polymorphisms as possible risk factors for disease or exposures. For all of the known functions of PON1, the level of PON1 enzyme is important and, in some cases, also the Q192R polymorphism. A simple high throughput two-substrate assay/analysis, plotting rates of diazoxon hydrolysis vs. paraoxon hydrolysis, provided both PON1 levels and functional Q192R phenotype/genotype. We have developed a new two-substrate assay/analysis protocol that provides PON1 status without use of toxic OP substrates. Factors were determined for inter-converting rates of hydrolysis of different substrates.

Biomarkers of sensitivity and exposure in Washington state pesticide handlers.

Organophosphate (OP) and N-methyl-carbamate (CB) insecticides are widely used in agriculture in the US and abroad. These compounds - which inhibit acetylcholinestersase (AChE) enzyme activity - continue to be responsible for a high proportion of pesticide poisonings among US agricultural workers. It is possible that some individuals may be especially susceptible to health effects related to OP/CB exposure. The paraoxonase (PON1) enzyme metabolizes the highly toxic oxon forms of some OPs, and an individual's PON1 status may be an important determinant of his or her sensitivity to these chemicals. This chapter discusses methods used to characterize the PON1 status of individuals and reviews previous epidemiologic studies that have evaluated PON1-related sensitivity to OPs in relation to various health endpoints. It also describes an ongoing longitudinal study among OP-exposed agricultural pesticide handlers who are participating in a recently implemented cholinesterase monitoring program in Washington State. This study will evaluate handlers' PON1 status as a hypothesized determinant of butyrylcholinesterase (BuChE) inhibition. Such studies will be useful to determine how regulatory risk assessments might account for differences in PON1-related OP sensitivity when characterizing inter-individual variability in risk related to OP exposure. Recent work assessing newer and more sensitive biomarkers of OP exposure is also discussed briefly in this chapter.

Engineering human PON1 in an E. coli expression system.

Expression and purification of recombinant human paraoxonase-1 (rHuPON1) from bacterial systems have proven elusive. Most systems for successful production of recombinant PON1 have relied on either eukaryotic expression in baculovirus or prokaryotic expression of synthetic, gene-shuffled rabbit-mouse-human PON1 hybrid molecules. We review here methods and protocols for the production of pure, native rHuPON1 using an E. coli expression system followed by conventional column chromatographic purification. The resulting rHuPON1 is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the organophosphorus (OP) compound diazoxon. Bacterially-derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that produces immunogenic complications when used as a therapeutic. The rHuPON1 should be useful for treating insecticide OP exposures and reducing risks of other diseases resulting from low PON1 status. The ease of mutagenesis in bacterial systems will also allow for the generation and screening of rHuPON1 variants with enhanced catalytic efficiencies against nerve agents and other OP compounds.

Identification and characterization of biomarkers of organophosphorus exposures in humans.

Over 1 billion pounds of organophosphorus (OP) chemicals are manufactured worldwide each year, including 70 million pounds of pesticides sprayed in the US. Current methods to monitor environmental and occupational exposures to OPs such as chlorpyrifos (CPS) have limitations, including low specificity and sensitivity, and short time windows for detection. Biomarkers for the OP tricresyl phosphate (TCP), which can contaminate bleed air from jet engines and cause an occupational exposure of commercial airline pilots, crewmembers and passengers, have not been identified. The aim of our work has been to identify, purify, and characterize new biomarkers of OP exposure. Butyrylcholinesterase (BChE) inhibition has been a standard for monitoring OP exposure. By identifying and characterizing molecular biomarkers with longer half-lives, we should be able to clinically detect TCP and OP insecticide exposure after longer durations of time than are currently possible. Acylpeptide hydrolase (APH) is a red blood cell (RBC) cytosolic serine proteinase that removes N-acetylated amino acids from peptides and cleaves oxidized proteins. Due to its properties, it is an excellent candidate for a biomarker of exposure. We have been able to purify APH and detect inhibition by both CPS and metabolites of TCP. The 120-day lifetime of the RBC offers a much longer window for detecting exposure. The OP-modified serine conjugate in the active site tryptic peptide has been characterized by mass spectrometry. This research uses functional proteomics and enzyme activities to identify and characterize useful biomarkers of neurotoxic environmental and occupational OP exposures.

Paraoxonase 1 (PON1) status and substrate hydrolysis.

Paraoxonase 1 (PON1) hydrolyzes a number of organophosphorus (OP) compounds including insecticides and nerve agents. The in vivo efficacy of PON1 to protect against a specific OP exposure depends on the catalytic efficiency of hydrolysis. The Q192R polymorphism affects the catalytic efficiency of hydrolysis of some substrates and not others. While PON1(R192) hydrolyzes paraoxon approximately 9-times as efficiently as PON1(Q192), the efficiency is insufficient to provide in vivo protection against paraoxon/parathion exposure. The two PON1(192) alloforms have nearly equivalent but higher catalytic efficiencies for hydrolyzing diazoxon (DZO) and provide equivalent in vivo protection against DZO exposures. On the other hand, PON1(R192) is significantly more efficient in hydrolyzing chlorpyrifos oxon (CPO) than PON1(Q192) and provides better protection against CPO exposure. Thus, for some exposures it is only the level of plasma PON1 that is important, whereas for others it is both plasma level and the PON1(192) alloform(s) present in plasma that are important. In no case is the plasma level of PON1 unimportant, provided that the catalytic efficiency is sufficient to protect against the exposure. Two-substrate enzyme assay/analysis protocols that reveal both PON1 plasma levels and PON1(192) phenotype (QQ; QR; RR) are designed to optimize the separation of PON1(192) phenotypes; however, they have not been optimized for evaluating in vivo rates of OP detoxication. This study describes the adaptation of a non-OP, two-substrate determination of PON1 status to the conversion of the PON1 status data to physiologically relevant rates of DZO and CPO detoxication. Conversion factors were generated for rates of hydrolysis of different substrates.

Ancient convergent losses of Paraoxonase 1 yield potential risks for modern marine mammals.

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.

Molecular insights into lipid-assisted Ca2+ regulation of the TRP channel Polycystin-2.

Polycystin-2 (PC2), a calcium-activated cation TRP channel, is involved in diverse Ca2+ signaling pathways. Malfunctioning Ca2+ regulation in PC2 causes autosomal-dominant polycystic kidney disease. Here we report two cryo-EM structures of distinct channel states of full-length human PC2 in complex with lipids and cations. The structures reveal conformational differences in the selectivity filter and in the large exoplasmic domain (TOP domain), which displays differing N-glycosylation. The more open structure has one cation bound below the selectivity filter (single-ion mode, PC2SI), whereas multiple cations are bound along the translocation pathway in the second structure (multi-ion mode, PC2MI). Ca2+ binding at the entrance of the selectivity filter suggests Ca2+ blockage in PC2MI, and we observed density for the Ca2+-sensing C-terminal EF hand in the unblocked PC2SI state. The states show altered interactions of lipids with the pore loop and TOP domain, thus reflecting the functional diversity of PC2 at different locations, owing to different membrane compositions.

Paraoxonase polymorphisms, haplotypes, and enzyme activity in Latino mothers and newborns.

Recent studies have demonstrated widespread pesticide exposures in pregnant women and in children. Plasma paraoxonase 1 (PON1) plays an important role in detoxification of various organophosphates. The goals of this study were to examine in the Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS) birth cohort of Latina mothers and their newborns living in the Salinas Valley, California, the frequencies of five PON1 polymorphisms in the coding region (192QR and 55LM) and the promoter region (-162AG, -909CG, and -108CT) and to determine their associations with PON1 plasma levels [phenylacetate arylesterase (AREase) ] and enzyme activities of paraoxonase (POase) and chlorpyrifos oxonase (CPOase) . Additionally, we report results of PON1 linkage analysis and estimate the predictive value of haplotypes for PON1 plasma levels. We found that PON1-909, PON1-108, and PON1(192) had an equal frequency (0.5) of both alleles, whereas PON1-162 and PON1(55) had lower variant allele frequencies (0.2) . Nearly complete linkage disequilibrium was observed among coding and promoter polymorphisms (p < 0.001) , except PON1(192) and PON1-162 (p > 0.4) . Children's PON1 plasma levels (AREase ranged from 4.3 to 110.7 U/mL) were 4-fold lower than their mothers' (19.8 to 281.4 U/mL) . POase and CPOase activities were approximately 3-fold lower in newborns than in mothers. The genetic contribution to PON1 enzyme variability was higher in newborns (R2 = 25.1% by genotype and 26.3% by haplotype) than in mothers (R2 = 8.1 and 8.8%, respectively) . However, haplotypes and genotypes were comparable in predicting PON1 plasma levels in mothers and newborns. Most of the newborn children and some pregnant women in this Latino cohort may have elevated susceptibility to organophosphate toxicity because of their PON1192 genotype and low PON1 plasma levels.

Paraoxonase 1 (PON1) Q192R genotypes and their interaction with smoking strongly increase atherogenicity and the Framingham risk score.

OBJECTIVE: Paraoxonase 1 (PON1) polymorphisms are associated with an increased susceptibility to cardiovascular disease. PON1 Q192R polymorphism (rs662) partially determine PON1 hydrolytic activity and protect against oxidation of LDL and HDL. This study aimed to delineate the association of PON1 status (functional 192 genotype and plasma activity levels) and atherogenicity in urbans residents aged 40 years or more. MATERIALS AND METHODS: Anthropometric data, lipid profiles, the atherogenic index of the plasma (AIP) and Framingham score risk were measured. Three kinetic assays were conducted to assay PON1 status using phenylacetate and 4-(chloromethyl)phenyl acetate as substrates. RESULTS: Smoking per se did not significantly impact the AIP but the interaction PON1 genotype by smoking significantly increased the AIP. In subjects with the RR genotype smoking increased the AIP index from (estimated mean ± SEM) -0.038 ± 0.039 to 0.224 ± 0.094. The QR genotype increased the Framingham risk index by around 1.3 points. Smoking by RR genotype carriers significantly increased the Framingham risk score (17.23 ± 2.04) as compared to smoking (13.00 ± 1.06) and non-smoking (7.79 ± 0.70) by QQ+QR genotype carriers. The interaction RR genotype by smoking was a more important predictor (odds ratio = 7.90) of an increased Framingham risk score (> 20) than smoking per se (odds ratio = 2.73). The interaction smoking by RR genotype carriers significantly increased triglycerides and lowered HDL cholesterol. CONCLUSION: Smoking per se has no (AIP) or a mild (Framingham risk score) effect on atherogenicity, while the interaction smoking by PON1 RR genotype has a clinically highly significant impact on atherogenicity.

Paraoxonase activity, but not haplotype utilizing the linkage disequilibrium structure, predicts vascular disease.

OBJECTIVE: The effects of paraoxonase (PON1) activity and of genetic variation in the PON1 promoter and coding region on carotid artery disease (CAAD) were investigated. METHODS AND RESULTS: We identified functional promoter polymorphisms and examined their effects in a cohort with and without CAAD. We used the full sequences in 23 white subjects to determine the linkage disequilibrium (LD) structure of the PON1 region and to direct the grouping of haplotypes for disease association testing. There are several discrete regions of the PON1 gene with strong local LD, but the useful levels of LD do not extend across the entire gene. Indeed, PON1-162/-108/55/192 haplotype did not predict additional variation in PON1 activities compared with the 4 genotypes separately. PON1 hydrolysis activity predicted CAAD status, but this was not attributable to the promoter or coding region polymorphisms or haplotype or to the effects of smoking or statin use on PON1 activity. CONCLUSIONS: PON1 does not have LD across the gene, and use of haplotypes in association studies should consider the LD structure. PON1 activity predicts CAAD, yet 4 functional polymorphisms do not. Additional investigations of genetic and environmental factors that influence PON1 activity as a risk factor for vascular disease are warranted.

Vitamin C and E intake is associated with increased paraoxonase activity.

OBJECTIVE: Paraoxonase (PON1), an esterase physically associated with high density lipoprotein, has been shown to inhibit atherogenic low density lipoprotein and high density lipoprotein oxidation. PON1 activity appears to be primarily under genetic control with some environmental modification and is a predictor of vascular disease. Vitamins C and E, dietary antioxidants, scavenge free-oxygen radical products that may depress PON1 activity. Therefore, we evaluated the relationship between dietary vitamin C and E intake and PON1 activity. METHODS AND RESULTS: The vitamin C and E intakes of male white subjects (n=189) were estimated by using a standardized food frequency survey. With covariates, vitamin C or E intakes were found to be significant positive predictors of PON1 activity for the hydrolysis of paraoxon and diazoxon with the use of linear regression. Smoking and use of statins were independent predictors of PON1 activity. CONCLUSIONS: PON1 activity, which is primarily genotype dependent, varies with antioxidant vitamins, cigarette smoking, and statin drug use. Because PON1 activity is a better predictor of vascular disease than is the currently described genetic variation in PON1, further studies of the environmental influences on PON1 activity and additional PON1 genetic variants are warranted.