RESUMEN
Human paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme that exhibits a broad substrate specificity. In addition to protecting against exposure to some organophosphorus (OP) pesticides by hydrolyzing their toxic oxon metabolites, PON1 is important in protecting against vascular disease by metabolizing oxidized lipids. Recently, PON1 has also been shown to play a role in inactivating the quorum sensing factor N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) of Pseudomonas aeruginosa. Native, untagged engineered recombinant human PON1 (rHuPON1) expressed in Escherichia coli and purified by conventional column chromatographic purification is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the OP compound diazoxon. The bacterially derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that can produce immunogenic complications when inappropriately glycosylated recombinant proteins are used as therapeutics. Previous studies have shown that the determination of PON1 status, which reveals both PON1(192) functional genotype and serum enzyme activity level, is required for a meaningful evaluation of PON1's role in risk of disease or exposure. We have developed a new two-substrate assay/analysis protocol that provides PON1 status without use of toxic OP substrates, allowing for use of this protocol in non-specialized laboratories. Factors were also determined for inter-converting rates of hydrolysis of different substrates. PON1 status also plays an important role in revealing changes in HDL-associated PON1 activities in male patients with Parkinson disease (PD). Immunolocalization studies of PONs 1, 2 and 3 in nearly all mouse tissues suggest that the functions of PONs 1 and 3 extend beyond the plasma and the HDL particle.
Asunto(s)
Arildialquilfosfatasa/metabolismo , Enfermedad , Exposición a Riesgos Ambientales/efectos adversos , Intoxicación por Organofosfatos , Animales , Arildialquilfosfatasa/genética , Arildialquilfosfatasa/uso terapéutico , Biomarcadores/metabolismo , Humanos , RiesgoRESUMEN
OBJECTIVE: Four recent studies report a genetic association of the paraoxonase locus with sporadic amyotrophic lateral sclerosis (ALS). We tested the hypothesis that this association correlates with functional changes in paraoxonase 1 (PON1, MIM 168820). METHODS: Sera from 140 ALS participants; 153 age-, race-, and sex-matched controls; and 30 matched CSF samples were tested for paraoxonase, diazoxonase, and arylesterase activities. Participants with ALS were genotyped using tagging single nucleotide polymorphisms across the PON locus. Survival data and enzyme activity were correlated with genotype. RESULTS: There was a trend toward increased paraoxonase activity in ALS compared with controls (mean control paraoxonase 701.9 +/- 469.7 U/L, mean ALS 792.5 +/- 574.1 U/L; p = 0.066 after correction) which correlated with increased frequency of the homozygous arginine (RR) variant of PON1(Q192R) (p = 0.004). There was no significant difference in PON1 protein levels, or arylesterase or diazoxonase activities. Organophosphate hydrolysis rates had no effect on ALS survival. CONCLUSIONS: Contrary to expectations, PON1 protein, paraoxonase, diazoxonase, and arylesterase activities were not reduced in amyotrophic lateral sclerosis (ALS). The increase in PON1(R192) frequency in ALS in our study supports previous genetic susceptibility studies. Our findings suggest that the influence of PON1 polymorphisms on ALS susceptibility is not due to reduced organophosphate hydrolysis.
Asunto(s)
Esclerosis Amiotrófica Lateral/sangre , Esclerosis Amiotrófica Lateral/enzimología , Arildialquilfosfatasa/sangre , Predisposición Genética a la Enfermedad/genética , Organofosfatos/metabolismo , Polimorfismo Genético/genética , Esclerosis Amiotrófica Lateral/genética , Arildialquilfosfatasa/análisis , Arildialquilfosfatasa/genética , Biomarcadores/análisis , Biomarcadores/sangre , Hidrolasas de Éster Carboxílico/análisis , Hidrolasas de Éster Carboxílico/sangre , Hidrolasas de Éster Carboxílico/genética , Estudios de Cohortes , Análisis Mutacional de ADN , Regulación hacia Abajo/genética , Activación Enzimática/genética , Femenino , Frecuencia de los Genes , Pruebas Genéticas , Genotipo , Humanos , Hidrólisis , Isoenzimas/sangre , Isoenzimas/genética , Masculino , Valor Predictivo de las Pruebas , Regulación hacia Arriba/genéticaRESUMEN
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.
Asunto(s)
Esterasas/genética , Polimorfismo Genético/genética , Regiones Promotoras Genéticas/genética , Arildialquilfosfatasa , Línea Celular , Esterasas/biosíntesis , Regulación de la Expresión Génica , Haplotipos , Humanos , Mutagénesis Sitio-Dirigida , Transfección , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Estenosis Carotídea/enzimología , Estenosis Carotídea/genética , Esterasas/genética , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos/genética , Arginina/genética , Arildialquilfosfatasa , Estudios de Casos y Controles , Genotipo , Glutamina/genética , Haplotipos , Humanos , Isoenzimas/genética , Leucina/genética , Masculino , Metionina/genética , Persona de Mediana Edad , Compuestos Organofosforados/metabolismo , Paraoxon/metabolismo , Fenotipo , Valor Predictivo de las Pruebas , Factores de RiesgoRESUMEN
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.
Asunto(s)
Esterasas/genética , Compuestos Organofosforados/toxicidad , Animales , Arildialquilfosfatasa , Cloropirifos/análogos & derivados , Cloropirifos/metabolismo , Esterasas/metabolismo , Frecuencia de los Genes , Genotipo , Cobayas , Humanos , Insecticidas/metabolismo , Hígado/enzimología , Ratones , Ratones Noqueados , Compuestos Organofosforados/metabolismo , Paraoxon/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Esterasas/genética , Esterasas/metabolismo , Leucina/genética , Metionina/genética , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos/genética , Arildialquilfosfatasa , Activación Enzimática/genética , Esterasas/sangre , Femenino , Tamización de Portadores Genéticos , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Polimorfismo Genético/genéticaRESUMEN
Susceptibility to organophosphorus (OP) insecticides and nerve agents is strongly influenced by genetic and developmental factors. A number of organophosphorothioate insecticides are detoxified in part via a two-step pathway involving bioactivation of the parent compound by the cytochrome P450 systems, then hydrolysis of the resulting oxygenated metabolite (oxon) by serum and liver paraoxonases (PON1). Serum PON1 has been shown to be polymorphic in human populations. The Arg192 isoform (PON1R192) of this HDL-associated protein hydrolyzes paraoxon (POX) at a high rate, while the Gln192 isoform (PON1Q192) hydrolyzes paraoxon at a low rate. The effect of the polymorphism is reversed for the hydrolysis of diazoxon (DZO), soman and particularly sarin. Phenylacetate is hydrolyzed at approximately the same rate by both PON1 isoforms and chlorpyrifos oxon (CPO) slightly faster by the PON1R192 isoform. In addition to the effect of the amino acid substitution on rates of toxicant hydrolysis, two other factors influence these rates. The expression of PON1 is developmentally regulated. Newborns have very low levels of PON1. Adult levels in rats and mice are reached at 3 weeks of age and in humans, sometime after 6 months of age. In addition, among individuals of a given genotype, there is at least a 13-fold difference in expression of PON1 that is stable over time. Dose/response experiments with normal mice injected with purified PON1 and with PON1 knockout mice have clearly demonstrated that the observed differences of in vitro rates of hydrolysis are significant in determining differential sensitivities to specific insecticides processed through the P450/PON1 pathway. Injection of purified rabbit PON1 protects mice from cholinesterase inhibition by chlorpyrifos (CPS) and CPO. Knockout mice are much more sensitive to CPO and DZO than are their PON1+/+ littermates or wild-type mice. A number of recent reports have also indicated that the PON1R192 isoform may be a risk factor for cardiovascular disease. Studies with PON1 knockout mice are also consistent with a role of PON1 in preventing vascular disease.
Asunto(s)
Esterasas/genética , Esterasas/metabolismo , Insecticidas/toxicidad , Síndromes de Neurotoxicidad/genética , Compuestos Organofosforados , Animales , Arildialquilfosfatasa , Humanos , Síndromes de Neurotoxicidad/enzimología , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Esterasas/genética , Compuestos Organofosforados/metabolismo , Animales , Arildialquilfosfatasa , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Catálisis , Cloropirifos/metabolismo , Inhibidores de la Colinesterasa/metabolismo , Diafragma/efectos de los fármacos , Diafragma/enzimología , Heterocigoto , Homocigoto , Humanos , Inactivación Metabólica , Insecticidas/metabolismo , Ratones , Ratones Noqueados , Paraoxon/metabolismo , Polimorfismo GenéticoRESUMEN
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.
Asunto(s)
Esterasas/genética , Esterasas/fisiología , Organofosfatos/farmacocinética , Animales , Arildialquilfosfatasa , Esterasas/sangre , Humanos , Inactivación Metabólica , Ratones , Ratones Noqueados , Organofosfatos/toxicidad , Reacción en Cadena de la Polimerasa , Polimorfismo GenéticoRESUMEN
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.
Asunto(s)
Esterasas/genética , Adolescente , Adulto , Arildialquilfosfatasa , Esterasas/sangre , Genotipo , Humanos , Persona de Mediana Edad , Polimorfismo GenéticoRESUMEN
Several organophosphorus insecticides and nerve agents are detoxified through the cytochrome P450/paraoxonase (PON1) pathway. PON1 is an HDL-associated enzyme encoded as a 355 amino acid protein in humans. The PON1 Arg192 isoform hydrolyzes paraoxon rapidly while the Gln192 isoform hydrolyzes this compound slowly. Both isoforms hydrolyze phenylacetate and chlorpyrifos oxon at approximately the same rate. We recently found that the effect of this polymorphism is dramatically reversed for sarin hydrolysis. The PON1 Arg192 isoform has virtually no sarinase activity while the Gln192 isoform has substantial activity. The Gln192 isoform also hydrolyzes diazoxon and soman faster than the Arg192 isoform. In addition to the large differences in rates of hydrolysis observed for some OP substrates by the two PON1 isoforms, there is also a large variability in serum PON1 concentrations that is stable over time between individuals. Thus, two factors govern the PON1 status of a given individual, the PON1 genotype as well as the amount of protein expressed from each allele. A two-dimensional enzyme analysis provides an excellent assessment of an individual's PON1 status, ie. the position 192 genotype as well as phenotype, or level of serum PON1 (Nature Genet 14:334-336). Do these interindividual differences in rates of substrate hydrolysis by PON1 reflect an individual's sensitivity or resistance to OP compounds processed through the P450/PON1 pathway? Injection of purified PON1 into mice clearly demonstrates the protective effect of having high serum levels of PON1 against toxicity by chlorpyrifos oxon or chlorpyrifos. Preliminary experiments with PON1 knockout mice, on the other hand, clearly demonstrate that low PON1 levels result in dramatically increased sensitivity to chlorpyrifos oxon. Attempts to express human PON1 in mice from constructs containing either of the human PON1 cDNA sequences were unsuccessful, despite the generation of the respective transgenic mice.
Asunto(s)
Sustancias para la Guerra Química/toxicidad , Esterasas/genética , Insecticidas/toxicidad , Polimorfismo Genético/genética , Acetilcolinesterasa/metabolismo , Alelos , Animales , Arildialquilfosfatasa , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Cloropirifos/análogos & derivados , Cloropirifos/toxicidad , Diafragma/efectos de los fármacos , Diafragma/enzimología , Humanos , Isoenzimas/efectos de los fármacos , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la PolimerasaRESUMEN
Many organophosphorus compounds (OPs) are potent cholinesterase inhibitors, accounting for their use as insecticides and, unfortunately, also as nerve agents. Each year there are approximately 3 million pesticide poisonings world-wide resulting in 220,00 deaths. In 1990, there were 1.36 million kg of chlorpyrifos, 4.67 million kg of diazinon and 1.23 million kg of ethyl parathion manufactured in the USA (data supplied by the USEPA). In addition to exposure risks during pesticide manufacturing, distribution and use, there are risks associated with the major international effort aimed at destroying the arsenals of nerve agents, including soman and sarin. The United States has pledged to destroy approximately 25,000 tons of chemical agents by the end of the decade. The high density lipoprotein (HDL)-associated enzyme paraoxonase (PON1) contributes significantly to the detoxication of several OPs (Fig. 1). The insecticides parathion, chlorpyrifos and diazinon are bioactivated to potent cholinesterase inhibitors by cytochrome P-450 systems. The resulting toxic oxon forms can be hydrolysed by PON1, which also hydrolyses the nerve agents soman and sarin (Fig. 1). PON1 is polymorphic in human populations and different individuals also express widely different levels of this enzyme. The Arg192 (R192) PON1 isoform hydrolyses paraoxon rapidly, while the Gln192 (Q191) isoform hydrolyses paraoxon slowly. Both isoforms hydrolyse chlorpyrifos-oxon and phenylacetate at approximately the same rate. The role of PON1 in OP detoxication is physiologically significant. Injected PON1 protects against OP poisoning in rodent model systems and interspecies differences in PON1 activity correlate well with observed median lethal dose (LD50) values. We report here a simple enzyme analysis that provides a clear resolution of PON1 genotypes and phenotypes allowing for a reasonable assessment of an individual's probable susceptibility or resistance to a given OP, extending earlier studies on this system. We also show that the effect of the PON1 polymorphism is reversed for the hydrolysis of diazoxon, soman and especially sarin, thus changing the view of which PON1 isoform is considered to be protective.
Asunto(s)
Esterasas/sangre , Esterasas/genética , Compuestos Organofosforados/metabolismo , Sarín/metabolismo , Soman/metabolismo , Arildialquilfosfatasa , Sustancias para la Guerra Química/metabolismo , Sustancias para la Guerra Química/farmacología , Resistencia a Medicamentos/genética , Esterasas/efectos de los fármacos , Homocigoto , Humanos , Isoenzimas , Compuestos Organofosforados/farmacología , Polimorfismo Genético , Sarín/farmacología , Soman/farmacología , América del SurRESUMEN
Paraxonase, an enzyme associated with the high density lipoprotein (HDL) particle, hydrolyzes paraoxon, the active metabolite of the insecticide parathion. Several studies have shown that paraxonase levels in humans have a distribution characteristic of two alleles, one with low activity and the other with high activity. Paraoxonase also has arylesterase activity, which does not exhibit activity polymorphism and can therefore serve as an estimate of enzyme protein. Although the ability of paraoxon to irreversibly inhibit lipoprotein lipase (LPL) has been exploited experimentally for many years, the role of plasma paraoxonase in lipoprotein metabolism is unknown. Seventy-two normal individuals were examined for paraoxonase genotypes, plasma paraoxonase and arylesterase activities, postheparin LPL and hepatic lipase (HL) activities, and lipoprotein levels to determine whether (1) paraoxonase activity or genotype determines lipoprotein levels via an effect on LPL or HL activity or (2) variation in LPL and HL activities determines HDL levels and indirectly affects paraoxonase activity and protein levels in plasma. In the entire group, paraoxonase activity was related to arylesterase activity and genotype. Whereas arylesterase activity was correlated with HDL cholesterol (HDL-C) and apolipoproteinA-I (apoA-I) levels, neither arylesterase nor paraoxonase was correlated with LPL or HL activity. Furthermore, LPL activity was positively correlated and HL inversely correlated with HDL cholesterol and apoA-I levels, whereas LPL was inversely correlated with triglyceride levels. The paraoxonase genotypes of the study group were 30 individuals homozygous for the low-activity allele, 38 heterozygotes, and 4 individuals homozygous for the high-activity allele. Paraoxonase genotype accounted for approximately .75 of the variation in paraoxonase activity. Paraoxonase activity was linearly related to arylesterase activity within each subgroup. No difference in either LPL or HL activity was seen as a function of paraoxonase genotype, nor were differences seen in plasma triglyceride or HDL-C by genotype by ANOVA. The relation between LPL and HL and components of HDL in the paraoxonase genotypic subgroups in general reflected the associations seen in the group as a whole. Multivariate analysis showed that LPL, HL, and arylesterase, a measure of paraoxonase mass, were independent predictors of HDL cholesterol, while paraoxonase genotype or activity was not. Thus, variation in LPL and HL appears to be significantly related to HDL cholesterol and apoA-I levels. The levels of HDL are a major correlate of paraoxonase protein levels, while paraoxonase genotype is the major predictor of plasma paraoxonase activity.
Asunto(s)
Esterasas/genética , Lipoproteína Lipasa/sangre , Lipoproteínas HDL/sangre , Adulto , Arildialquilfosfatasa , Esterasas/sangre , Femenino , Heterocigoto , Homocigoto , Humanos , Masculino , Análisis MultivarianteRESUMEN
Apolipoprotein J (apoJ)-containing high-density lipoproteins (HDL), isolated from human plasma by immunoaffinity chromatography, are associated with apoAI and a protein of approximately 44 kDa. In order to advance our understanding of apoJ's role in the vasculature, a comprehensive investigation was performed to identify and characterize this 44-kDa protein and to study its interaction with apoJ. The 44-kDa protein, a monomeric glycoyslated polypeptide, was identified by N-terminal sequencing as serum paraoxonase. Paraoxonase exists in two oxidation states: one contains all free cysteines while the other has one disulfide bond between Cys42 and Cys284. Northern analysis of eight human tissues shows paraoxonase message present only in the liver. The majority of apoJ/paraoxonase-HDL are 90-140 kDa; however, not all of the plasma paraoxonase is associated with apoJ. The specificity of the apoJ/paraoxonase interaction, inferred by the constant mole ratio of the two proteins in affinity-purified apoJ-HDL, is confirmed in direct binding assays. For purified proteins, there is more than a 5-fold increase in the apparent affinity of apoJ for immobilized paraoxonase as the paraoxonase coating concentration is increased from 0.5 to 2.0 micrograms/mL. Both oxidation states of paraoxonase bind to apoJ with equal affinity. Our data combined with other evidence suggest that the plasma link of apoJ with paraoxonase will be implicated as a predictor of vascular damage.
Asunto(s)
Esterasas/sangre , Glicoproteínas/sangre , Chaperonas Moleculares , Secuencia de Aminoácidos , Arildialquilfosfatasa , Clusterina , ADN Complementario/genética , Disulfuros/química , Esterasas/química , Esterasas/genética , Esterasas/metabolismo , Glicoproteínas/metabolismo , Humanos , Isoenzimas , Hígado/enzimología , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Unión Proteica , Conformación Proteica , ARN Mensajero/análisis , Análisis de SecuenciaRESUMEN
Human and rabbit paraoxonases/arylesterases were purified to homogeneity by chromatographic and gel electrophoretic/isofocusing procedures coupled with activity stains. N-terminal and peptide sequence analysis suggested retention of the secretion signal sequence and allowed design of oligonucleotide probes. The probes were used to isolate a 1294-bp rabbit paraoxonase cDNA clone, which, in turn, was used to isolate three human cDNA clones. Comparison of rabbit and human protein and cDNA sequences indicated a high degree of sequence conservation (approximately 85% identity) and verified that paraoxonase retains its signal sequence (except for the N-terminal Met). The rabbit cDNA encodes a protein of 359 amino acids and the human a protein of 355 amino acids. In situ hybridization demonstrated, as expected, that the paraoxonase gene maps to the long arm of human chromosome 7. Arginine at position 192 specifies high activity paraoxonase and glutamine low activity human paraoxonase. Variation in protein levels explains the variation of enzyme activity observed within a genetic class. Toxicity studies showed that raising rat plasma paraoxonase levels by i.v. administration of partially purified rabbit paraoxonase protected animals against cholinesterase inhibition by paraoxon and chlorpyrifos oxon. Protection correlated with the relative rates of hydrolysis of these two compounds.
Asunto(s)
Compuestos Organofosforados/farmacocinética , Monoéster Fosfórico Hidrolasas/fisiología , Polimorfismo Genético/genética , Secuencia de Aminoácidos , Animales , Arildialquilfosfatasa , Secuencia de Bases , Clonación Molecular , ADN/genética , Humanos , Inactivación Metabólica , Datos de Secuencia Molecular , Mapeo Peptídico , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Conejos , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad por SustratoRESUMEN
Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.
Asunto(s)
Monoéster Fosfórico Hidrolasas/sangre , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Arildialquilfosfatasa , Cromatografía en Agarosa , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida/métodos , Activación Enzimática , Humanos , Hidrólisis , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/química , Conejos , Coloración y Etiquetado , Especificidad por SustratoRESUMEN
Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme [Costa et al. (1990) Toxicol. Appl. Pharmacol. 103, 66-76]. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined by (1) gas-phase sequencing of the intact protein and (2) peptide fragments from lysine and arginine digests. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. A clone was isolated and sequenced, and contained a 1294-bp insert encoding an open reading frame of 359 amino acids. Northern blot hybridization with RNA isolated from various rabbit tissues indicated that paraoxonase mRNA is synthesized predominately, if not exclusively, in the liver. Southern blot experiments suggested that rabbit paraoxonase is coded by a single gene and is not a family member of closely related genes. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Each of the human clones appeared to be polyadenylated at a different site, consistent with the absence of the canonical polyadenylation signal sequence. Of potential significance with respect to the paraoxonase polymorphism, the derived amino acid sequence from one of the partial human cDNA clones differed at two positions from the full-length clone. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue [Furlong et al. (1991) Biochemistry (preceding paper in this issue)]. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.
Asunto(s)
ADN/aislamiento & purificación , Monoéster Fosfórico Hidrolasas/sangre , Monoéster Fosfórico Hidrolasas/genética , Señales de Clasificación de Proteína/genética , Adulto , Secuencia de Aminoácidos , Animales , Arildialquilfosfatasa , Secuencia de Bases , Clonación Molecular , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Señales de Clasificación de Proteína/sangre , Señales de Clasificación de Proteína/aislamiento & purificación , ARN Mensajero/análisis , Conejos , Alineación de SecuenciaRESUMEN
Paraoxon and chlorpyrifos-oxon, the active metabolites of the organophosphorus insecticides parathion and chlorpyrifos, respectively, are hydrolyzed by an "A"-esterase, paraoxonase, which is present in the sera of several mammalian species. In this study, we investigated whether levels of serum paraoxonase activity in laboratory animals can influence the in vivo toxicity of paraoxon and chlorpyrifos-oxon. Paraoxonase was found to be 7-fold higher in rabbit serum than in rat serum. The dose of paraoxon required to produce similar signs of toxicity and similar degrees of cholinesterase inhibition in rats and rabbits (0.5 and 2.0 mg/kg, respectively) differed by 4-fold. Paraoxonase was then purified from rabbit serum and 8.35 units was injected in the tail veins of rats, increasing the peak hydrolytic activity of rat serum by 9-fold toward paraoxon and by 50-fold toward chlorpyrifos-oxon. The increase in serum paraoxonase/chlorpyrifos-oxonase activity was long-lasting, with a 2- and 10-fold increase, respectively, still present after 24 hr. Thirty minutes following enzyme injection, rats were challenged with an acute dose of paraoxon or chlorpyrifos-oxon given by the intravenous, intraperitoneal, dermal, or oral route. Cholinesterase activities were measured in plasma, red blood cells, brain, and diaphragm after 4 hr. Rats pretreated with paraoxonase exhibited less inhibition of cholinesterase than vehicle-treated controls following identical doses of paraoxon, particularly when the organophosphate was given iv or dermally. A very high degree of protection, particularly toward brain and diaphragm cholinesterase, was provided by paraoxonase pretreatment in animals challenged with chlorpyrifos-oxon by all routes. These results indicate that levels of serum paraoxonase activity can affect the toxicity of paraoxon and chlorpyrifos-oxon.
Asunto(s)
Cloropirifos/análogos & derivados , Paraoxon/toxicidad , Monoéster Fosfórico Hidrolasas/sangre , Animales , Arildialquilfosfatasa , Cloropirifos/administración & dosificación , Cloropirifos/toxicidad , Colinesterasas/análisis , Masculino , Paraoxon/administración & dosificación , Monoéster Fosfórico Hidrolasas/fisiología , Ratas , Ratas EndogámicasRESUMEN
Human serum plasma paraoxonase/arylesterase exhibits a genetic polymorphism for the hydrolysis of paraoxon. One allelic form of the enzyme hydrolyzes paraoxon slowly with a low turnover number and the other(s) hydrolyzes paraoxon rapidly with a high turnover number. Chlorpyrifos-oxon, the active metabolite of the insecticide chlorpyrifos (Dursban), is also hydrolyzed by plasma arylesterase/paraoxonase. A specific assay for measuring hydrolysis of this compound is described. This assay is not subject to interference by the esterase activity of serum albumin. The Km for chlorpyrifos-oxon hydrolysis was 75 microM. Hydrolysis was inhibited by phenyl acetate, EDTA, and organic solvents. Enzyme activity required calcium ions and was stimulated by sodium chloride. Hydrolysis was optimized by using methanol instead of acetone to dissolve substrate. Unlike the multimodal distribution of paraoxonase, the distribution of chlorpyrifos-oxonase activity failed to show clear multimodality. An improvement in the assay for hydrolysis of paraoxon by plasma arylesterase/paraoxonase was achieved by elimination of organic solvents. Plotting chlorpyrifos-oxonase activity vs paraoxonase activity for a human population using the new assay conditions provided an excellent resolution of low activity homozygotes from heterozygotes for this allele. A greater than 40-fold difference in rates of chlorpyrifosoxon hydrolysis observed between rat (low activity) and rabbit sera (high activity) correlated well with the reported large differences in LD50 values for chlorpyrifos in these two animals, consistent with an important role of serum paraoxonase in detoxification of organophosphorus pesticides in vivo.
Asunto(s)
Cloropirifos/metabolismo , Paratión/metabolismo , Monoéster Fosfórico Hidrolasas/sangre , Espectrofotometría/métodos , Acetona/farmacología , Animales , Arildialquilfosfatasa , Cloruro de Calcio/farmacología , Cloropirifos/análogos & derivados , Cloropirifos/toxicidad , Hidrólisis , Cinética , Dosificación Letal Mediana , Metanol/farmacología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Conejos , Ratas , Cloruro de Sodio/farmacologíaRESUMEN
Plasma paraoxonase is a polymorphic enzyme that hydrolyzes paraoxon, the neurotoxic, active metabolite of the insecticide parathion. This enzyme is specified by at least two alleles with frequencies of about .7 and .3 among Caucasoid populations. A specific assay was developed that measured the activity of human plasma paraoxonase without interference from serum albumin which contributes significantly to the hydrolytic breakdown of paraoxon at the high pH values used in many previous assays. There was an 11-fold variation in paraoxonase activities, and the population distribution was at least bimodal. However, this specific assay did not improve the discrimination between the three genetic classes: (1) homozygotes for the low-activity allele, (2) heterozygotes, and (3) homozygotes for the high-activity allele. Chlorpyrifos oxon--the neurotoxic metabolite of the organophosphorus insecticide chlorpyrifos (Dursban)--was hydrolyzed by the same plasma fraction that hydrolyzed paraoxon. There was only four- to fivefold variability in enzyme activity, and the population distribution was unimodal. Homozygotes for low paraoxonase activity ranged over almost the entire spectrum of chlorpyrifos oxonase activity. Possible differences in susceptibility to chlorpyrifos toxicity therefore are unlikely to be predicted by the paraoxonase genotype alone. The ratio of paraoxonase over that of chlorpyrifos oxonase provided an excellent method for genetic typing of the paraoxonase polymorphism, as did the substitution of phenylacetate for chlorpyrifos as the substrate.