Paraoxonase 1 promoter and coding region polymorphisms in Parkinson's disease.
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Adult↗
Biomedical subjects
C E Furlong
Publications and source records attributed to C E Furlong.
Effects of 5' regulatory-region polymorphisms on paraoxonase-gene (PON1) expression.
Human HDL-associated paraoxonase (PON1) hydrolyzes a number of toxic organophosphorous compounds and reduces oxidation of LDLs and HDLs. These properties of PON1 account for its ability to protect against pesticide poisonings and atherosclerosis. PON1 also hydrolyzes a number of lactone and cyclic-carbonate drugs. Among individuals in a population, PON1 levels vary widely. We previously identified three polymorphisms in the PON1 regulatory region that affect expression levels in cultured human hepatocytes. In this study, we determined the genotypes of three regulatory-region polymorphisms for 376 white individuals and examined their effect on plasma-PON1 levels, determined by rates of phenylacetate hydrolysis. The -108 polymorphism had a significant effect on PON1-activity level, whereas the -162 polymorphism had a lesser effect. The -909 polymorphism, which is in linkage disequilibrium with the other sites, appears to have little or no independent effect on PON1-activity level in vivo. Other studies have found that the L55M polymorphism in the PON1-coding region is associated with differences in both PON1-mRNA and PON1-activity levels. The results presented here indicate that the L55M effect of lowered activity is not due to the amino acid change but is, rather, largely due to linkage disequilibrium with the -108 regulatory-region polymorphism. The codon 55 polymorphism marginally appeared to account for 15.3% of the variance in PON1 activity, but this dropped to 5% after adjustments for the effects of the -108 and Q192R polymorphisms were made. The -108C/T polymorphism accounted for 22.8% of the observed variability in PON1-expression levels, which was much greater than that attributable to the other PON1 polymorphisms. We also identified four sequence differences in the 3' UTR of the PON1 mRNA.
3' Untranslated Regions↗
Polymorphisms in the human paraoxonase (PON1) promoter.
Paraoxonase (PON1) is a protein component of high-density lipoprotein (HDL) particles that protects against oxidative damage to both low-density lipoprotein and HDL and detoxifies organophosphorus pesticides and nerve agents. A wide range of expression levels of PON1 among individuals has been observed. We examined the promoter region of PON1 for genetic factors that might affect PON1 activity levels. We conducted a deletion analysis of the PON1 promoter region in transient transfection assays and found that cell-type specific promoter elements for liver and kidney are present in the first 200bp upstream of the coding sequence. Sequence analysis of DNA from a BAC clone and a YAC clone identified five polymorphisms in the first 1000 bases upstream of the coding region at positions -108, -126, -162, -832 and -909. Additionally, the promoter sequences of two individuals expressing high levels of PON1 and two individuals expressing low levels of PON1 were analysed. The two polymorphisms at -126 and -832 had no apparent effect on expression level in the reporter gene assay. The polymorphisms at position -909, -162 (a potential NF-I transcription factor binding site) and -108 (a potential SP1 binding site) each have approximately a two-fold effect on expression level. The expression level effects of the three polymorphisms appear not to be strictly additive and may depend on context effects.
Aryldialkylphosphatase↗
Analysis of paraoxonase (PON1) L55M status requires both genotype and phenotype.
Paraoxonase (PON1) is tightly associated with high-density lipoprotein particles and is believed to contribute to the prevention of atherosclerosis by metabolizing oxidized lipids. PON1 also hydrolyses the bioactive oxon forms of organophosphorus pesticides such as parathion, diazinon and chlorpyrifos. Two common polymorphisms have been identified in the coding sequence of human PON1: L55M and R192Q. Several previous studies have found that the presence of the PON1R192 allele raises the risk of cardiovascular disease while others found no correlation. The studies, however, have focused on the genotype of PON1 and not the expression level of the protein. We found that the PON1 expression level in plasma, as determined by the rates of paraoxon and diazoxon hydrolysis, varies widely among individuals and within a genotype. Previous studies found that individuals having Met at PON155 have lower levels of both PON1 mRNA and activity. In this study, we determined the plasma activity levels of PON1 and examined the relationships between PON155 genotype and PON1 level. As with PON1192, we found considerable overlap in activity among the PON155 genotypes. Of the 317 individuals whose PON1 status was determined in this study, 48.9% were PON1Q192 homozygotes. Analysis of the PON1QQ192 population showed that while the average PON1 activity (diazoxon hydrolysis) was 12266 U/L for PON1LL55 and 7777 U/L for PON1MM55, a given PONMM55 individual could have more than twice the activity of a PON1LL55 individual. PON1 status, which includes PON1 level as well as PON1192 genotype, may be a better predictor for cardiovascular disease or organophosphate susceptibility than PON1 genotype alone.
Aged↗
Catalytic efficiency determines the in-vivo efficacy of PON1 for detoxifying organophosphorus compounds.
Human paraoxonase (PON1) is a polymorphic, high-density lipoprotein (HDL)-associated esterase that hydrolyzes the toxic metabolites of several organophosphorus (OP) insecticides and nerve agents. The activity polymorphism is determined by a Gln/Arg (Q/R) substitution at position 192. Injection of purified PON1 protects animals from OP poisoning. In the present study, we investigated the in-vivo function of PON1 for detoxifying organophosphorus insecticides in PON1-knockout mice that were challenged via dermal exposure with diazoxon, diazinon and paraoxon. PON1-knockout mice were extremely sensitive to diazoxon. Doses (2 and 4 mg/kg) that caused no cholinesterase (ChE) inhibition in wild-type mice were lethal to the knockout mice, which also showed slightly increased sensitivity to the parent compound diazinon. Surprisingly, these knockout mice did not show increased sensitivity to paraoxon. In-vitro assays indicated that the PON1R192 isoform hydrolyzed diazoxon less rapidly than did the PON1Q192 isoform. In-vivo analysis, where PON1-knockout mice received the same amount of either PON1(192) isoform via intraperitoneal (i.p.) injection 4 h prior to exposure, showed that both isoforms provided a similar degree of protection against diazoxon, while PON1R192 conferred better protection against chlorpyrifos-oxon than PON1Q192. Injection of purified rabbit PON1 or either human PON1(192) isoform did not protect PONI-knockout mice from paraoxon toxicity, nor did over-expression of the human PON1R192 transgene in wild-type mice. Kinetic analysis of the two human PON1(192) isoforms revealed that the catalytic efficiency (Vmax/Km) determines the in-vivo efficacy of PON1 for organophosphorus detoxication. The results indicate that PON1 plays a major role in the detoxication of diazoxon and chlorpyrifos oxon but not paraoxon.
Animals↗
PON1 status and neurologic symptom complexes in Gulf War veterans.
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Animals↗
Paraoxonase (PON1) phenotype is a better predictor of vascular disease than is PON1(192) or PON1(55) genotype.
The paraoxonase (PON1) PON1-Q192R and PON1-L55M polymorphisms have been inconsistently associated with vascular disease. Plasma PON1 activity phenotypes vary markedly within genotypes and were, therefore, expected to add to the informativeness of genotype for predicting vascular disease. The case-control sample included 212 age- and race-matched men (mean age 66.4 years). The 106 carotid artery disease (CAAD) cases had >80% carotid stenosis, and the 106 controls had <15%. Two PON1 substrate hydrolysis rates (paraoxon [POase] and diazoxon [DZOase]) were significantly lower in cases than in controls and were significant predictors of CAAD by use of logistic regression (POase, P=0.005; DZOase, P=0.019). DZOase predicted vascular disease independently of lipoprotein profile, high density lipoprotein subfractions, apolipoprotein A-I, and smoking. PON1-192 and PON1-55 genotypes or haplotypes did not predict case-control status unless the activity phenotype was also included as a predictor by use of logistic regression. When phenotype was included as a predictor, PON1-192 and PON1-55 genotypes or combined haplotypes were significant predictors (P<0.05). In conclusion, examining PON1-192 and/or PON1-55 genotypes alone may mistakenly lead to the conclusion that there is no role of PON1 in CAAD. These results support the benefit of a "level crossing" approach that includes intervening phenotypes in the study of complexly inherited disease.
Aged↗
The PON1 gene and detoxication.
It has been assumed since its discovery that serum paraoxonase (PON1) plays a major role in the detoxication of specific organophosphorus compounds. It was also assumed that individuals with low PON1 activity would be more susceptible to paraoxon/parathion poisoning than individuals with higher PON1 activity. Evidence supporting this hypothesis was provided by injection of rabbit PON1 into rodents. Injected PON1 protected against paraoxon toxicity in rats and chlorpyrifos oxon toxicity in mice. The recent availability of PON1 knockout mice has provided an in vivo system with which one can more closely examine the role of PON1 in detoxication. PON1 knockout mice demonstrated dramatically increased sensitivity to chlorpyrifos oxon and diazoxon and moderately increased sensitivity to the respective parent compounds. The PON1 knockout mutation also resulted in the elimination of liver PON1 activity, accounting for the dramatic increase in sensitivity to chlorpyrifos oxon and diazoxon. Totally unexpected was our finding that the PON1 knockout mice were not more sensitive to paraoxon. This was particularly surprising in light of the earlier enzyme injection experiments. Differences in the relative catalytic efficiencies of rabbit vs. mouse PON1 for the specific oxon forms explain these observations. Mouse PON1 has good catalytic efficiency for the hydrolysis of diazoxon and chlorpyrifos oxon, but a poor efficiency for paraoxon hydrolysis relative to rabbit PON1. The human PON1Q192 isoform has a catalytic efficiency similar to that of mice, whereas the human PON1R192 isoform has a much better catalytic efficiency, predicting that individuals expressing high levels of the PONIR192 isoform may have increased resistance to paraoxon toxicity.
Animals↗
The role of paraoxonase (PON1) in the detoxication of organophosphates and its human polymorphism.
In human populations, serum paraoxonase (PON1) exhibits a substrate dependent polymorphism. The Arg192 isoform hydrolyzes paraoxon rapidly but diazoxon, soman and especially sarin slowly. On the other hand, the Gln192 isoform hydrolyzes paraoxon slowly, but diazoxon, soman and sarin more rapidly than the Arg192 isoform. Our experiments with a mouse model system have convincingly shown that PON1 plays a major role in the detoxication of organophosphate (OP) compounds processed through the P450/PON1 pathway. Recent studies have also shown that PON1 plays an important role in the metabolism of oxidized lipid compounds. Currently, there is an effort underway to identify genes and polymorphisms that play an important role in 'environmental susceptibility'. The PON1 polymorphism has been cited as a prime example of such a genetic polymorphism. The advent of the polymerase chain reaction (PCR) for DNA amplification with improvements, modifications and automation has provided a very convenient way to do individual genotyping. It is tempting to set up large scale PCR analyses of populations to determine individuals at risk for environmental exposures affected by the PON1 polymorphism. In fact, a number of such studies have already been carried out in examining the relationship of the PON1 polymorphism to vascular disease. We advocate the use of a high throughput two-dimensional enzyme assay that provides both PON1 genotype and phenotype (PON1 status). The high level of variation of gene expression within each genetic class in humans, together with our animal model studies indicate that it is very important to determine PON status as opposed to PON1 genotype alone. Experiments in rats and mice have shown that injection of PON1 purified from rabbit serum by the i.v., i.p. or i.m. route, significantly increases PON1 activities in rodents' plasma. Under these conditions, the acute toxicity (assessed by the degree of acetylcholinesterase inhibition) of paraoxon and chlorpyrifos oxon is significantly decreased, compared to control animals. Protection is maximal when PON1 is administered before the OPs, but still occurs when PON1 is utilized as a post-exposure treatment. Furthermore, protection by PON1 is also provided toward the parent compound chlorpyrifos. Pon1-knockout mice display a much greater sensitivity to chlorpyrifos oxon toxicity than wild mice. However, the acute toxicity of guthion, which is not a substrate for PON1, does not differ between knockout and wild mice. These observations underline the importance of considering both genetic variability of enzyme isoform as well as enzyme level (PON1 status) and the developmental time course of appearance of PON1 in developing risk assessment models.
Animals↗
Recommended nomenclature system for the paraoxonases.
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Amino Acid Sequence↗
Symposium overview: the role of genetic polymorphism and repair deficiencies in environmental disease.
A symposium of this title was presented at the 37th Annual Meeting of the Society of Toxicology held in Seattle, Washington during March of 1998. The symposium focused on heritable variations in metabolism, DNA replication, and DNA repair that may predispose humans to environmental diseases. Human metabolic, replication, and repair enzymes function in protective roles. Metabolic enzymes are protective because they detoxify a stream of chemicals to which the body is exposed. Replication and repair enzymes are also protective; they function to maintain the integrity of the human genome. Polymorphisms in the genes that code for some of these enzymes are known to give rise to variations in their protective functions. For example, functional polymorphisms of the N-acetyltransferases, paraoxonases, and microsomal epoxide hydrolases vary in their capacity to metabolize environmental chemicals. Specific isoforms of the N-acetyltransferases and microsomal epoxide hydrolases are increasingly associated with incidences of cancer attributable to exposure to these chemicals. Thus, maintenance of cellular-growth homeostasis, normally and in the face of environmental challenge, is dependent on an inherited assortment of metabolic isoforms. Since replication and repair are also protective cellular functions, and since mutations in genes that code for these functions are associated with tumorigenesis, one can reasonably speculate that common functional polymorphisms of replication and repair enzymes may also impart susceptibility to environmental disease.
Animals↗
Determination of paraoxonase (PON1) status requires more than genotyping.
Human serum paraoxonase (PON1) is associated with high density lipoprotein (HDL) particles. This enzyme is involved in the metabolism of oxidized lipids and also plays a major role in the metabolism and detoxication of insecticides processed through the cytochrome P450/PON1 pathway. An Arg/Gln (R/Q) substitution at position 192 determines a substrate dependent activity polymorphism. In addition to the effect of the amino acid substitution on rates of hydrolysis of different substrates, there is a large interindividual variability in the amount of PON1 protein in sera that is stable over time. Recently, a number of reports based solely on PON1 genotyping have suggested that in some populations, the PON(R192) allele may be a risk factor for coronary artery disease. Another report notes an increased risk of the PON(R192) allele for Parkinson's disease. We report here the development of a two-dimensional, microtitre plate reader-based enzyme analysis that provides a high-throughput assessment of PON1 status. Population distribution plots of diazoxonase versus paraoxonase activities provides PON1 phenotype and an accurate inference of PON1 genotype. Both are important parameters for determining an individual's PON1 status. The analysis also provides PON1 allele frequencies for specific populations.
Adolescent↗
Construction of biosensors using a gold-binding polypeptide and a miniature integrated surface plasmon resonance sensor.
Surface plasmon resonance (SPR) biosensors were constructed on miniature integrated sensors. Recognition elements were attached to the sensor surface using a gold-binding repeating polypeptide. Biosensors with fluorescyl groups attached to their surfaces were functional for at least 1 month of daily use with little decrease in response to the binding of an anti-fluorescyl monoclonal antibody. The coupling of protein A to the gold-binding polypeptide on the sensor surface enabled the biosensor to detect the binding of antibodies to the protein A and provided a sensor with convertible specificity. The system described herein provides a simple and rapid approach for the fabrication of highly specific, durable, portable and low cost SPR-based biosensors.
Amino Acid Sequence↗
Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis.
Serum paraoxonase (PON1) is an esterase that is associated with high-density lipoproteins (HDLs) in the plasma; it is involved in the detoxification of organophosphate insecticides such as parathion and chlorpyrifos. PON1 may also confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids present in oxidized low-density lipoproteins (LDLs). To study the role of PON1 in vivo, we created PON1-knockout mice by gene targeting. Compared with their wild-type littermates, PON1-deficient mice were extremely sensitive to the toxic effects of chlorpyrifos oxon, the activated form of chlorpyrifos, and were more sensitive to chlorpyrifos itself. HDLs isolated from PON1-deficient mice were unable to prevent LDL oxidation in a co-cultured cell model of the artery wall, and both HDLs and LDLs isolated from PON1-knockout mice were more susceptible to oxidation by co-cultured cells than the lipoproteins from wild-type littermates. When fed on a high-fat, high-cholesterol diet, PON1-null mice were more susceptible to atherosclerosis than their wild-type littermates.
Animals↗
Simulated dermal contamination with capillary samples and field cholinesterase biomonitoring.
The extensive international use of organophosphorus compounds (OP) results in numerous acute intoxications each year. OPs inhibit acetylcholinesterase, the enzyme responsible for breaking down the neurotransmitter acetylcholine. The World Health Organization recognizes cholinesterase (ChE) biomonitoring as a preventive measure against OP overexposure. The aim of this study was to determine if dermal OP contamination could interfere with current field ChE biomonitoring assays, which use a fingerstick blood sample. In this study we also sought to determine if high levels of a plasma enzyme, A-esterase, could protect ChE from inhibition by hydrolyzing environmentally generated oxons potentially present in a fingerstick sample. A heparinized venous blood sample was collected from a volunteer. Erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (PChE) activities were measured using a field-based colorimetric cholinesterase kit. ChE dose-response curves were constructed by allowing 10-microliters blood samples to contact environmentally realistic levels of OP thioate and oxon for 10 s. An inhibition threshold could not be established for PChE when exposed to oxon within the time necessary to perform a fingerstick analysis. AChE was also inhibited by trace amounts of oxon consistent with previously reported environmental levels. These findings suggest that the reliability of field-based biomonitoring results is limited if OP residues remain on a skin surface at the time of sample collection. A-esterase's role in protecting ChE activity was investigated using capillary and venous blood from 30 unexposed individuals. Baseline ChE activities were measured, as were individual A-esterase activities using paraoxon, diazoxon, and phenylacetate as substrates. Results were then compared to ChE activities measured after 10 s of contact with an environmentally realistic amount of OP, containing 1% oxon. Both ChE activities were significantly inhibited, with capillary values being significantly more inhibited than their venous counterparts. However, no protective effect could be associated between the degree of A-esterase activity and the subsequent level of ChE inhibition observed in an individual's blood. These results suggest that (1) if there is any uncertainty about OP skin contamination, venous blood would be a more appropriate specimen to employ when using field ChE biomonitoring kits--it is collected in larger volumes and has essentially no direct contact to dermal surfaces; and (2) A-esterase activity demonstrates no protective effect against ChE inhibition upon a blood droplet's brief contact with an OP residue containing traces of oxon.
Acetylcholinesterase↗
Paraoxonase (PON1) gene in mice: sequencing, chromosomal localization and developmental expression.
Serum paraoxonase hydrolyses the toxic metabolites of several organophosphorus insecticides, as well as the nerve agents soman and sarin. We have previously shown that elevated serum paraoxonase levels protect mice against organophosphate toxicity. In the present study, we determined the cDNA sequence and chromosomal location of the mouse paraoxonase gene, as well as its developmental expression in mice and rats. The mouse cDNA encodes a protein of 355 amino acids and shows 81% identity with the human sequence. In situ hybridization demonstrated that the mouse paraoxonase gene maps to chromosome 6, a region conserved with the paraoxonase region of chromosome 7q21-22 in humans. Serum paraoxonase activities toward three substrates, paraoxon, chlorpyrifos-oxon and diazoxon, were very low at birth and increased with age reaching adult levels at 20 days in mice and 25 days in rats. The increase of serum paraoxonase activity in developing animals correlates well with the increased resistance to organophosphate poisoning that has been reported in previous studies.
Age Factors↗
Structural organization of the human PON1 gene.
Serum paraoxonase/arylesterase (PON) is an "A" esterase found in the HDL2 fraction of mammalian sera closely associated with apolipoproteins A1 and J. This enzyme hydrolyzes the active metabolites (oxons) of several organophosphate (OP) insecticides as well as the P-F bond of the nerve agents soman and sarin. PON also destroys biologically active, multioxygenated phospholipids. Two factors result in large individual variations in PON serum levels, a substrate-dependent activity polymorphism and large individual differences in PON protein levels that are stable over time. Animal model studies indicate that PON activity levels are likely to play a major role in determining sensitivity to OPs. The arg192 PON isoform appears to be a risk factor in coronary artery disease. We report here the characterization of a 28-kb contig encompassing 300 bp of 5' sequence, the entire coding region, and 2 kb of 3'-flanking sequence of the PON gene. The structural portion of the paraoxonase protein is encoded by nine exons that form the primary transcript through the use of typical splice donor and acceptor sites. DNA sequences of the regions surrounding all the coding exons have been determined. A polymorphic CA repeat is located in intron 4.
Aryldialkylphosphatase↗
Expression of apolipoprotein E inhibits aggregation of the C-terminal fragments of beta-amyloid precursor protein.
An important role of apolipoprotein E in the amyloidogenesis of Alzheimer's disease is suggested by an accumulation of apolipoprotein E in beta-amyloid plaques and a genetic association between Alzheimer's disease and one of the allelic variants (APOE4) of apolipoprotein E. Overexpression of a C-terminal region of beta-amyloid precursor protein brings about aggregation of the C-terminal fragments in COS cells. This COS cell culture system was used to study effects of apolipoprotein E on aggregation of the C-terminal fragments. When both apolipoprotein E and the C-terminal fragments were overexpressed in COS cells, Western blot analyses revealed significant inhibition of aggregation of the C-terminal fragments. No significant differences between apolipoprotein E3 and E4 in the inhibitory activities were found by this method. Apolipoprotein E may inhibit formation of amyloid fibrils.
Alzheimer Disease↗