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.

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

ABSTRACT 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.

Human valacyclovir hydrolase/biphenyl hydrolase-like protein is a highly efficient homocysteine thiolactonase.

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

Proteomic analysis of adducted butyrylcholinesterase for biomonitoring organophosphorus exposures.

Organophosphorus (OP) compounds include a broad group of toxic chemicals such as insecticides, chemical warfare agents and antiwear agents. The liver cytochromes P450 bioactivate many OPs to potent inhibitors of serine hydrolases. Cholinesterases were the first OP targets discovered and are the most studied. They are used to monitor human exposures to OP compounds. However, the assay that is currently used has limitations. The mechanism of action of OP compounds is the inhibition of serine hydrolases by covalently modifying their active-site serine. After structural rearrangement, the complex OP inhibitor-enzyme is irreversible and will remain in circulation until the modified enzyme is degraded. Mass spectrometry is a sensitive technology for analyzing protein modifications, such as OP-adducted enzymes. These analyses also provide some information about the nature of the OP adduct. Our aim is to develop high-throughput protocols for monitoring OP exposures using mass spectrometry.

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.

Additional Common Polymorphisms in the PON Gene Cluster Predict PON1 Activity but Not Vascular Disease.

Background. Paraoxonase 1 (PON1) enzymatic activity has been consistently predictive of cardiovascular disease, while the genotypes at the four functional polymorphisms at PON1 have not. The goal of this study was to identify additional variation at the PON gene cluster that improved prediction of PON1 activity and determine if these variants predict carotid artery disease (CAAD). Methods. We considered 1,328 males in a CAAD cohort. 51 tagging single-nucleotide polymorphisms (tag SNPs) across the PON cluster were evaluated to determine their effects on PON1 activity and CAAD status. Results. Six SNPs (four in PON1 and one each in PON2/3) predicted PON1 arylesterase (AREase) activity, in addition to the four previously known functional SNPs. In total, the 10 SNPs explained 30.1% of AREase activity, 5% of which was attributable to the six identified predictive SNPs. We replicate rs854567 prediction of 2.3% of AREase variance, the effects of rs3917510, and a PON3 haplotype that includes rs2375005. While AREase activity strongly predicted CAAD, none of the 10 SNPs predicting AREase predicted CAAD. Conclusions. This study identifies new genetic variants that predict additional PON1 AREase activity. Identification of SNPs associated with PON1 activity is required when evaluating the many phenotypes associated with genetic variation near PON1.

Biomarkers of organophosphorus (OP) exposures in humans.

There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of "biomarker proteins" that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS.

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.

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.

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.

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.

PON1 status of farmworker mothers and children as a predictor of organophosphate sensitivity.

The objective was to determine PON1 status as a predictor for organophosphorus insecticide sensitivity in a cohort of Latina mothers and newborns from the Salinas Valley, California, an area with high levels of organophosphorus insecticide use. PON1 status was established for 130 pregnant Latina women and their newborns using a high-throughput two substrate activity/analysis method which plots rates of diazoxon (DZO) hydrolysis against rates of paraoxon (PO) hydrolysis. Arylesterase activity (AREase) was determined using phenylacetate as a substrate, allowing comparison of PON1 levels across PON1192 genotypes in mothers and children. Phenylacetate hydrolysis is not affected by the Q192R polymorphism. Among newborns, levels of PON1 (AREase) varied by 26-fold (4.3-110.7 U/ml) and among mothers by 14-fold (19.8-281.4 U/ml). On average, children's PON1 levels were four-fold lower than the mothers' PON1 levels (P<0.001). Average PON1 levels in newborns were comparable with reported hPON1 levels in transgenic mice expressing human PON1Q192 or PON1R192, allowing for prediction of relative sensitivity to chlorpyrifos oxon (CPO) and DZO. The predicted range of variability in sensitivity of mothers and children in the same Latino cohort was 65-fold for DZO and 131 to 164-fold for CPO. Overall, these findings indicate that many of the newborns and some of the mothers in this cohort would be more susceptible to the adverse effects of specific organophosphorus pesticide exposure due to their PON1 status. Of particular concern are exposures of pregnant mothers and newborns with low PON1 status.

TagSNP analyses of the PON gene cluster: effects on PON1 activity, LDL oxidative susceptibility, and vascular disease.

Paraoxonase 1 (PON1) activity is consistently predictive of vascular disease, although the genotype at four functional PON1 polymorphisms is not. To address this inconsistency, we investigated the role of all common PON1 genetic variability, as measured by tagging single-nucleotide polymorphisms (tagSNPs), in predicting PON1 activity for phenylacetate hydrolysis, LDL susceptibility to oxidation ex vivo, plasma homocysteine (Hcy) levels, and carotid artery disease (CAAD) status. The biological goal was to establish whether additional common genetic variation beyond consideration of the four known functional SNPs improves prediction of these phenotypes. PON2 and PON3 tagSNPs were secondarily evaluated. Expanded analysis of an additional 26 tagSNPs found evidence of previously undescribed common PON1 polymorphisms that affect PON1 activity independently of the four known functional SNPs. PON1 activity was not significantly correlated with LDL oxidative susceptibility, but genotypes at the PON1(-108) promoter polymorphism and several other PON1 SNPs were. Neither PON1 activity nor PON1 genotype was significantly correlated with plasma Hcy levels. This study revealed previously undetected common functional PON1 polymorphisms that explain 4% of PON1 activity and a high rate of recombination in PON1, but the sum of the common PON1 locus variation does not explain the relationship between PON1 activity and CAAD.

Role of paraoxonase (PON1) status in pesticide sensitivity: genetic and temporal determinants.

Individual differences in detoxication capacities for specific organophosphorous (OP) compounds are due largely to differences in catalytic efficiency or abundance of the HDL-associated enzyme, paraoxonase (PON1). First, we provide evidence that children less than 2 years of age represent a particularly susceptible population for OP exposure due to low abundance of PON1 and variable onset of plasma PON1 activity. Second, we describe studies examining the neurotoxic effects of chronic, low-level OP pesticide exposure in mice. PON1 knockout (PON1(-/-)) and wild-type mice were exposed chronically (PN4 to PN21) to low levels of chlorpyrifos oxon (CPO). Endpoints included cholinesterase activity, histopathology, gene expression, and behavior. Even at PN4, when PON1 levels were low in wild-type mice, PON1(-/-) mice were more sensitive to inhibition of brain cholinesterase by CPO. At PN22, and persisting as long as 4 months, chronic developmental exposure to 0.18 mg/kg/d or 0.25 mg/kg/d CPO resulted in perinuclear vacuolization of cells in a discrete area of the neocortex and irregular distribution of neurons in the cortical plate, with an increase in the number of affected cells at 0.25mg/kg/d. Third, we describe a transgenic mouse model in which human transgenes encoding either hPON1Q192 or hPON1R192 were expressed at equal levels in place of mouse PON1. The developmental onset of expression followed the mouse time course and was identical for the two transgenes, allowing these mice to be used to assess the importance of the Q192R polymorphism during development. Adult mice expressing hPON1R192 were significantly more resistant than hPON1Q192 mice to CPO toxicity. Our studies indicate that children less than 2 years old, especially those homozygous for PON1Q192, would be predicted to be particularly susceptible to CPO toxicity.

The correlation of paraoxonase (PON1) activity with lipid and lipoprotein levels differs with vascular disease status.

Paraoxonase (PON1) is an HDL-associated enzyme. Low PON1 activity predicts vascular disease status and is a more reliable predictor of vascular disease than are functional PON1 genotypes. There is evidence that the relationship of PON1 to vascular disease is, in part, due to its antioxidant activity. However, the physical relationship of PON1 with HDL and the existence of cholesterol pathway regulatory elements at the PON1 locus suggest a further relationship of PON1 with lipoproteins, which may contribute to its role in vascular disease. We investigated the relationship of PON1 activity and genotype to lipid-related traits in 91 Caucasian men with severe carotid artery disease and 184 without vascular disease who were not on lipid-lowering medications. Prior studies of PON1 relationship to lipids have not stratified by disease status.. We found that PON1 activity was correlated with HDL traits in controls and with LDL- and VLDL-related traits in cases. We hypothesize differences in the joint regulation of PON1 and lipoproteins in cases and controls.

Toxicity of chlorpyrifos and chlorpyrifos oxon in a transgenic mouse model of the human paraoxonase (PON1) Q192R polymorphism.

OBJECTIVES: The Q192R polymorphism of paraoxonase (PON1) has been shown to affect hydrolysis of organophosphorus compounds. The Q192 and R192 alloforms exhibit equivalent catalytic efficiencies of hydrolysis for diazoxon, the oxon form of the pesticide (DZ). However, the R192 alloform has a higher catalytic efficiency of hydrolysis than does the Q192 alloform for chlorpyrifos oxon (CPO), the oxon form of the pesticide chlorpyrifos (CPS). The current study examined the relevance of these observations for in-vivo exposures to chlorpyrifos and chlorpyrifos oxon. METHODS: Using a transgenic mouse model we examined the relevance of the Q192R polymorphism for exposure to CPS and CPO in vivo. Transgenic mice were generated that expressed either human PON1Q192 or PON1R192 at equivalent levels, in the absence of endogenous mouse PON1. Dose-response and time course experiments were performed on adult mice exposed dermally to CPS or CPO. Morbidity and acetylcholinesterase (AChE) activity in the brain and diaphragm were determined in the first 24 h following exposure. RESULTS: Mice expressing PON1Q192 were significantly more sensitive to CPO, and to a lesser extent CPS, than were mice expressing PON1R192. The time course of inhibition following exposure to 1.2 mg/kg CPO revealed maximum inhibition of brain AChE at 6-12 h, with PON1R192, PON1Q192, and PON1 mice exhibiting 40, 70 and 85% inhibition, respectively, relative to control mice. The effect of PON1 removal on the dose-response curve for CPS exposure was remarkably consistent with a PBPK/PD model of CPS exposure. CONCLUSION: These results indicate that individuals expressing only the PON1Q192 allele would be more sensitive to the adverse effects of CPO or CPS exposure, especially if they are expressing a low level of plasma PON1Q192.