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.
The organophosphate cholinesterase inhibitor paraoxon is hydrolysed by serum paraoxonase/arylesterase. A genetic polymorphism of paraoxonase (PON) activity which determines high versus low paraoxon hydrolysis in human populations, may determine sensitivity to parathion poisoning. We demonstrate that arginine at position 192 specifies high activity PON whereas a glutamine specifies the low activity variant. Allele-specific probes or restriction enzyme analysis of amplified DNA allow for the genotyping of individuals. PON maps to chromosome 7q21-22, proximal to the cystic fibrosis gene, in agreement with previous genetic linkage studies.
Therapies that raise levels of HDL, which is thought to exert atheroprotective effects via effects on endothelium, are being examined for the treatment or prevention of coronary artery disease (CAD). However, the endothelial effects of HDL are highly heterogeneous, and the impact of HDL of patients with CAD on the activation of endothelial eNOS and eNOS-dependent pathways is unknown. Here we have demonstrated that, in contrast to HDL from healthy subjects, HDL from patients with stable CAD or an acute coronary syndrome (HDL CAD ) does not have endothelial antiinflammatory effects and does not stimulate endothelial repair because it fails to induce endothelial NO production. Mechanistically, this was because HDL CAD activated endothelial lectin-like oxidized LDL receptor 1 (LOX-1), triggering endothelial PKCβII activation, which in turn inhibited eNOS-activating pathways and eNOS-dependent NO production. We then identified reduced HDL-associated paraoxonase 1 (PON1) activity as one molecular mechanism leading to the generation of HDL with endothelial PKCβII-activating properties, at least in part due to increased formation of malondialdehyde in HDL. Taken together, our data indicate that in patients with CAD, HDL gains endothelial LOX-1-and thereby PKCβII-activating properties due to reduced HDL-associated PON1 activity, and that this leads to inhibition of eNOS-activation and the subsequent loss of the endothelial antiinflammatory and endothelial repair-stimulating effects of HDL.
Although pesticide use is widespread, little is known about potential adverse health effects of in utero exposure. We investigated the effects of organophosphate pesticide exposure during pregnancy on fetal growth and gestational duration in a cohort of low-income, Latina women living in an agricultural community in the Salinas Valley, California. We measured nonspecific metabolites of organophosphate pesticides (dimethyl and diethyl phosphates) and metabolites specific to malathion (malathion dicarboxylic acid), chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphoro-thioate], and parathion (4-nitrophenol) in maternal urine collected twice during pregnancy. We also measured levels of cholinesterase in whole blood and butyryl cholinesterase in plasma in maternal and umbilical cord blood. We failed to demonstrate an adverse relationship between fetal growth and any measure of in utero organophosphate pesticide exposure. In fact, we found increases in body length and head circumference associated with some exposure measures. However, we did find decreases in gestational duration associated with two measures of in utero pesticide exposure: urinary dimethyl phosphate metabolites [β adjusted = -0.41 weeks per log 10 unit increase; 95% confidence interval (CI), -0.75--0.02; p = 0.02], which reflect exposure to dimethyl organophosphate compounds such as malathion, and umbilical cord cholinesterase (β adjusted = 0.34 weeks per unit increase; 95% CI, 0.13-0.55; p = 0.001). Shortened gestational duration was most clearly related to increasing exposure levels in the latter part of pregnancy. These associations with gestational age may be biologically plausible given that organophosphate pesticides depress cholinesterase and acetylcholine stimulates contraction of the uterus. However, despite these observed associations, the rate of preterm delivery in this population (6.4%) was lower than in a U.S. reference population.
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.
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.
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