Original article 183

PON1 status of farmworker mothers and children as a predictor of organophosphate sensitivity
Clement E. Furlonga,*, Nina Hollandb,*, Rebecca J. Richtera, Asa Bradmanb, Alan Hob and Brenda Eskenazib
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 highthroughput 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 (4.3110.7 U/ml) and among mothers by 14-fold (19.8281.4 U/ml). On average, childrens 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 65fold 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. Pharmacogenetics and Genomics c 16:Lippincott Williams & Wilkins.
Pharmacogenetics and Genomics 2006, 16:183190 Keywords: children, chlorpyrifos, diazinon, Latino cohort, paraoxonase 1 (PON1), PON1 status, pregnancy
a Departments of Genome Sciences and Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington and bCenter for Childrens Environmental Health, School of Public Health, University of California, Berkeley, California, USA
Correspondence and requests for reprints to Clement Furlong, University of Washington, Division of Medical Genetics, Box 357720, Seattle, WA 98195-7720, USA Tel: + 543 1193; fax: + 543 3050; e-mail: clem@u.washington.edu Sponsorship: This study was supported by 2 P01 ES009605, ES11387, ES09883, P30 ES01896, EPA-R82670901-5, P60 MD00222, and NIEHS ES09601/EPA: RD-83170901. The contents of this paper are solely the responsibility of the authors and do not necessarily represent official views of the NIH, or the EPA. Received 26 July 2005 Accepted 13 October 2005

Introduction

Recent biological and ambient monitoring data have indicated widespread organophosphate pesticide exposures to the US population, including adults, pregnant women, children and fetuses [19]. In some cases, these exposures may exceed health-based reference levels [10 11]. Although many uses, including residential applications, of chlorpyrifos (CPS) and diazinon (DZ) were recently restricted by agreements with registrants [12,13], agricultural uses are still widespread. Organophosphate exposures at high doses have profound effects, primarily on the central nervous system [14], and there is growing evidence in animals and humans to suggest that chronic low level exposure may affect neurodevelopment [5,1517]. The young of many species are more susceptible to organophosphate toxicity than adults [1822]. For example, the maximum tolerated dose (MTD) of CPS in
7-day-old (PND7) rats is approximately 7.7% of the MTD in adult animals [22]. One factor contributing to the increased sensitivity in newborns is that levels of paraoxonase 1/arylesterase (PON1), a key organophosphate detoxifying enzyme, are three- to four-fold lower than in adults [2327]. Even among adults, the levels of PON1 can vary by at least 13-fold [28]. Human PON1 enzyme, a high-density lipoprotein(HDL) associated esterase, is encoded as a 355 amino acid protein by the PON1 gene on chromosome 7q21.3 22.1 [29] with only its initiator methionine residue removed before incorporation into HDL particles [30,31]. In humans, a Q192R polymorphism affects the catalytic efficiency of hydrolysis of some organophosphate substrates [28,32,33], including chlorpyrifos oxon (CPO), the toxic metabolite of CPS [34]. The characterization of all 28 TagSNPs accounts for only 28% of the variance in PON1 levels (G.P. Jarvik, personal communication), much of which is attributable to a C-108T polymorphism in an Sp1 binding site in the 50 regulatory region [3537].
The first two authors contributed equally to this work.

c 1744-Lippincott Williams & Wilkins
Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Pharmacogenetics and Genomics 2006, Vol 16 No 3
Simple biochemical principles dictate that rates of detoxication of substrates are dependent on enzyme levels. Thus, when considering the effects of genetic variability on sensitivity to CPS/CPO exposures, both the quantity (level) of PON1 as well as the quality (Q192 versus R192) of PON1 must be considered. In other cases where the catalytic efficiency of hydrolysis of the two PON1192 alloforms is equivalent [e.g. for diazoxon (DZO) hydrolysis], it is the level of PON1 that is important [34]. Li et al. [38] introduced the term PON1 status to include both PON1 level and functional PON1192 genotype (Q/Q; Q/R; R/R) [38]. In this method, PON1 status is determined with a simple, high-throughput two-substrate assay and analysis where rates of DZO hydrolysis are plotted against rates of PO hydrolysis using serum or nonEDTA preserved plasma samples. This method, validated in adults, provides a functional determination of the PON1192 alloform(s) present in plasma, as well as the level of an individuals plasma PON1 [3941], both important in modulating exposures to organophosphorus compounds as well as other risks associated with PON1 status. The lower PON1 levels associated with PON1M55 [42] are primarily attributable to linkage disequilibrium with the inefficient promoter polymorphism T-108 [35]. The development of animal model systems has provided important insights into the role of PON1 in detoxifying specific organophosphorus compounds. Injection of purified rabbit PON1 into rats increased resistance to paraoxon (PO) exposure [43,44] and, more significantly, to CPO exposure [44] because rabbit PON1 hydrolyses CPO very rapidly [45]. These observations were confirmed and extended using mice, which required much less purified PON1 for injections and were more amenable to genetic manipulation [34,38,46]. Injected PON1 protected against CPS/CPO exposures when injected 30 min or 24 h before exposure or up to 3 h post exposure [47]. These experiments provided convincing evidence that high levels of PON1 protected against CPS/CPO exposures. The increase in resistance was most dramatic against CPO exposures [34]. The generation of PON1 knockout mice by Shih et al. [46] provided a model with which to examine the consequence of the absence of plasma PON1 on resistance to organophosphate exposures. The PON1 null mice were found to have dramatically increased sensitivity to CPO [46] and DZO exposure [34], and less noticeably increased sensitivity to the respective parent compounds CPS [46,47] and DZ [34]. It was surprising to find that the PON1 null mice did not have increased sensitivity to PO exposure [34]. Injection of purified human PON1Q192 or PON1R192 reconstituted PON1 activity in the serum of the PON1

PON1 status and predicted organophosphate sensitivity Furlong et al. 185
another 14% had other jobs in agriculture, including packing shed, nursery and greenhouse work. Overall, 82% of subjects had agricultural workers living in their homes during pregnancy. Additional information about exposure and associations with health outcomes is reported elsewhere [7,8,16,51]. All study protocols were approved by both the University of California, Berkeley and the University of Washington human-subject review processes. Informed consent was obtained for all subjects.
Biological sample collection and processing
Blood was collected from mothers at the time of their glucose tolerance test (26 2.3 weeks gestation) and in the hospital immediately before or after delivery. Umbilical cord blood was collected by delivery room staff once the baby was safely delivered. Heparinized whole blood was centrifuged, and divided into plasma, buffy coats and red blood cells, and stored at 801C. Processed plasma samples were stored at 801C before being shipped on dry ice to the University of Washington, Seattle, where they were also stored at 801C until analysis of enzyme activity. We first compared PON1 activities in 25 mothers at two time points (26 weeks gestation and delivery). PON1 activities were highly correlated between the two time points for all three measured activities of PON1 (r = 0.7, 0.8, 1.0, P < 0.0001 for AREase, CPOase and DZOase, respectively). The ranges for the two measurements were comparable. Therefore, in subsequent samples, we measured PON1 activities in blood collected at one time point only (26 weeks gestation).
Determination of PON1 status
samples and has been shown to identify individuals with mutations in the PON1 gene [41]. Although this plot suggests that PON1R192 homozygotes have lower rates of DZO hydrolysis than heterozygotes and PON1Q192 homozygotes, this is not the case, as the position 192 alloforms have equivalent catalytic efficiencies for DZO hydrolysis [34]. However, the PON1R192 alloform is more sensitive to inhibition by the high salt concentration intentionally used in this assay to resolve the three PON1192 phenotypes. The distributions and descriptive statistics of PON1 status and enzyme levels in mothers and their newborns were analysed by STATA 8.0 [54].

Results

PON1 status
PON1 status (functional PON1192 genotype and plasma level) was determined by the method developed and validated on adult populations by Richter and Furlong [39]. This method provides an accurate determination of functional PON1192 genotype through the use of a twosubstrate enzyme kinetic analysis. PON1 enzyme activities in plasma of mothers and children were measured with three different substrates, including paraoxon (POase), diazoxon (DZOase) and phenylacetate (AREase), according to published protocols [3941,52]. Because phenylacetate hydrolysis is not affected by the Q192R polymorphism and has been shown to correspond with PON1 levels determined by immunological methods [29,53], AREase activity was used for measurement of PON1 levels across genotypes. For PON1 status determination, rates of DZO hydrolysis were plotted against rates of PO hydrolysis for each mother and newborn child (cord blood) in the study. This analysis separates the population into three distinct groups, individuals functionally homozygous for PON1Q192, PON1Q/R192 heterozygotes and individuals functionally homozygous for PON1R192. The accuracy of the PON1192 functional genotype determination has been verified by polymerase chain reaction (PCR) analysis of more than 2000 adult
Figure 1 shows the PON1 status for mothers and newborns (cord blood) as determined by the plot of rates of hydrolysis of DZO versus PO. This plot clearly resolves the three functional PON1192 phenotypes (verified by PCR analysis) for the mothers. However, a number of data points for newborns with lower activity values (22 out of 130 or approximately 17%) were not resolved by this analysis and could only be accurately assigned by genotyping the Q192R polymorphism. However, the PON1 status plot provided relative PON1 levels for each of the 22 subjects. This analysis also shows the effects of independent allelic expression in the heterozygotes (including the newborns) with some individuals expressing more of one PON1192 alloform than the other. Genotyping alone does not provide this information. Several points are worth noting from this analysis: (i) there is a large variability of PON1 activity values within each PON1192 genotype, both for mothers and newborns; (ii) both groups of homozygotes fall closely to the trend line; and (iii) the heterozygote values vary considerably from the trend line (trend line not shown), consistent with the concept of independent cis regulation of each PON1 allele (Fig. 1).
Range of plasma PON1 levels
Figure 2 shows PON1 levels across genotypes for mothers and newborns estimated by the AREase activity. Figure 3 shows the effects of the PON1192 polymorphism on rates of paraoxon hydrolysis across genotypes for mothers and newborns, and provides an excellent example of why substrates whose rates of hydrolysis are affected by the PON1192 polymorphism should not be used to compare levels across position 192 genotypes. Rates of paraoxon hydrolysis are significantly lower for Q/Q homozygotes and heterozygotes than for R/R homozygotes. The AREase activity in mothers ranged from a low of 19.8 U/ml to a high of 281.4 U/ml (14-fold) and, in newborns, from 4.3110.7 U/ml (26-fold) (Fig. 2). The range of AREase activity (PON1 levels) from the lowest newborn to the highest mother was 4.3281.4 U/ml (65-fold). The mean PON1 levels, as measured by AREase, were similar across PON1192 genotypes for all mothers (Q/Q = 152

Fig. 1

Fig. 3

CordQQ MaternalQQ QQ

CordQR MaternalQR
CordRR MaternalRR QQ mother
Paraoxonase, U/l 1000 2000

Diazoxonase, U/l

QR RR Q192R genotype 3500 Paraoxonase, U/l Arylesterase child, U/ml Arylesterase, U/ml 100 200

QQ child

QR mother

QR child

RR mother

RR child

PON1 status plot for mothers and newborns, determined as described in the Methods section. Open circles, data points for PON1Q192 homozygous mothers; closed circles, data points for PON1Q192 homozygous newborns; closed squares, data points for heterozygous mothers; open squares, data points for heterozygous newborns; open triangles, data points for PON1R192 homozygous mothers; closed triangles, data points for PON1R192 homozygous newborns. Individual data points for paraoxonase activities in mothers (solid circles) and newborns (open circles) for each PON1192 genotype as indicated. Means are indicated by crossbars.

Fig. 4 Fig. 2

QQ mother

QQ child Q192R genotype

Arylesterase mother, U/ml 300
Individual data points for arylesterase activities (AREase) in mothers (solid circles) and newborns (open circles) for each PON1192 genotype as indicated. Means are indicated by the crossbars.
Plot of arylesterase activities (AREase) of newborn/mother pairs (r = 0.47, P = 0.013).
newborns had on average four-fold less plasma PON1 than mothers, individual levels in mother-child pairs were modestly correlated (r = 0.47, P = 0.013) (Fig. 4).
Estimation of the range of sensitivity to organophosphate exposure
U/ml, Q/R = 144 U/ml and R/R = 152 U/ml). The mean PON1 levels were also similar across genotypes in newborns (Q/Q = 31 U/ml, Q/R = 36 U/ml and R/R = 43 U/ml), with the newborn PON1R192 homozygotes having somewhat higher average AREase activity than the PON1Q192 homozygotes and the newborn heterozygotes expressing intermediate levels. However, these AREase activity differences between genotype groups were not statistically significant (P = 0.13). Although
Because both PON1192 alloforms hydrolyse DZO with the same catalytic efficiency [34], these data predict a range of sensitivity to DZO exposure of 26-fold in newborns and 14-fold in mothers, with a range of 65-fold from the most sensitive newborn to the most resistant mother. An average four-fold difference in sensitivity to DZO exposure is predicted between mothers and newborns.

PON1 status and predicted organophosphate sensitivity Furlong et al. 187
Estimation of the range of sensitivity to CPO exposure is more complex, due to the different catalytic efficiencies of the two PON192 alloforms for CPO hydrolysis. However, reasonable estimates of relative sensitivity can be made by taking advantage of data generated in the mouse model system, where the mouse PON1 gene was replaced with either human hPON1Q192 or human hPON1R192 and colonies were established that expressed these alloforms at the same levels in plasma. Transgenic mice expressing hPON1Q192 were found to be 2- to 2.5fold as sensitive to CPO exposure compared to mice expressing hPON1R192 [48], due to the higher catalytic efficiency of human PON1R192 for hydrolysis of CPO [30]. This two- to 2.5-fold difference in sensitivity, taken together with the 65-fold difference in plasma AREase levels yield an estimated 131164-fold range in CPO sensitivity between the PON1Q/Q192 homozygous newborn with the lowest PON1 level and the PON1R/R192 homozygous mother with the highest PON1 level.
borns had higher PON1 levels than some of the adults. Thus, the individuals predicted to be highly sensitive to organophosphate exposure include most of the newborns as well as some of the mothers. The studies reported here confirm earlier observations of lower PON1 activities in neonates compared to adults [2327,56] and are also consistent with a recent report where neonates had 2.6 4.6-fold lower PON1 levels, as assessed by the AREase assay, compared to mothers, in three ethnic groups in New York City [23]. Among the many activities of the multifunctional HDLassociated enzyme PON1, hydrolysis of DZO and CPO is important in providing protection against exposure to DZ/ DZO and CPS/CPO. Organophosphate exposures are appropriately considered as mixed exposures to the parent compounds and their oxon forms because most, if not all, exposures include oxon residues [57,58]. Because the rate of cholinesterase inactivation by CPO is at least three orders of magnitude higher than that of its parent compound (CPS) [59], a very small percentage of oxon form in an exposure is significant. To date, most of the animal model studies with genetically modified mice have examined CPO/CPS exposures [34,38,4648,60], although some recent studies were carried out on DZ/DZO exposures in PON1 null mice and PON1 null mice injected with each of the purified human PON1192 alloforms [34]. The results obtained in these earlier studies have provided important insights and predictions with respect to individual variability in sensitivity to CPO/CPS and DZ/DZO exposures. Li et al. [34] examined the relative in-vivo catalytic efficiencies of hPON1Q192 and hPON1R192 in PON1 null mice reconstituted with purified human PON1R192 or PON1Q192. They found that under in-vivo physiological conditions, injection of either PON1192 alloform provided equivalent protection against DZO exposure, whereas injected human PON1R192 provided significantly better protection against CPO exposure than did PON1Q192. A more recent study by Cole et al. [48], using transgenic strains of mice that expressed either PON1Q192 or PON1R192 at nearly equivalent levels, verified the results of Li et al. [34] from the enzyme injection studies in the PON1 null mice. The mice expressing PON1R192 were approximately two- to 2.5-fold more resistant to CPO exposure than were the PON1 null mice. Mice expressing PON1Q192 were nearly as sensitive as the PON1 null mice to CPO exposures. The IC50 value (exposure level for 50% inhibition) for CPO inhibition in the hPONQ192 transgenic mice was approximately 1.1 mg/kg dermal exposure versus approximately 2.2 mg/kg in the hPON1R192 mice. The levels of PON1 in the hPON1 humanized transgenic mice are relevant to the studies reported here. The transgenic mice expressed levels of

Discussion

To our knowledge, this is the first study where PON1 status [39,41,52] was determined for a large cohort of mothers and their newborns using the two-substrate assay. By plotting rates of DZO hydrolysis against rates of PO hydrolysis, the three Q192R phenotypes were clearly separated in the mothers and provided the relative PON1 levels for each individual in this population. However, we found that for 17% of the newborns, with low PON1 levels, PCR analysis was required to assign position 192 genotypes, although the PON1 status analysis did provide their cord blood PON1 levels. Although some of the earlier studies examined only PON1 status with the two substrate diazoxon/paraoxon assay/analysis, this study included a measurement of rates of phenylacetate hydrolysis (AREase), allowing comparison of PON1 levels across PON1192 genotypes. Another study examining the relationship of PON1 levels, exposure and head circumference in a large cohort of 404 births also made use of AREase activity to determine PON1 levels [55], representing a major improvement over the many studies that examined PON1 genotypes alone [49]. They noted a relationship between low PON1 levels, exposure and smaller head circumference. The AREase values provided an estimate of the relative sensitivity to organophosphorus compounds whose in-vivo catalytic efficiencies of hydrolysis are not affected by the PON1192 polymorphism (e.g. DZO). The range of AREase values in the mothers was very similar to the range observed in another study of Hispanic farm workers in Washington State where a 13-fold variability was observed [28]. The large range of AREase values observed in this study in the newborns (26-fold) predicts a broad variability in sensitivity to organophosphate exposure even among newborns. Surprisingly, some of the new-
hPON1 of 3045 U/ml AREase (corrected for baseline AREase activity in PON1 null mice) [48], which are comparable to the average levels of PON1 in the Latino newborns described in the present study. Cole et al. [24] examined the time course of appearance of PON1 in the plasma of individual children and also in mice expressing the two human PON1192 alloforms under control of the human 50 and 30 regulatory sequences. In children, PON1 reached plateau levels at 624 months of age whereas expression of human hPON1 in mice under control of the human PONregulatory region followed the mouse time course of PON1 appearance, peaking at PND21. Moser et al. [62] observed that, although AREase activity in rats peaked at 34 weeks of age as observed by Li et al. [61], resistance to CPS exposure continued to increase up to at least 90 days, corresponding to a continued increase in plasma carboxylesterase activity. Although there is very little carboxylesterase activity in human serum, individual variation in carboxylesterase levels in human liver microsomes has been reported [63]. Levels of PON1 activities reported in other studies provide insights into the range of sensitivities expected. The hydrolytic activities in plasma of the wild-type C57Bl/6J mice used in our earlier studies were approximately 1800 U/l for chlorpyrifos oxonase (CPOase) and approximately 3500 U/l for diazoxonase (DZOase) [60]. These activities are in the low range relative to those reported for human populations. The ranges of reported activities in another adult Hispanic population were U/l for CPOase and U/l for DZOase [28]. It is worth noting that PON1 null mice are approximately ten-fold more sensitive to DZO [34] and CPO [46] exposure than wild-type mice. Based on what is known about the catalytic efficiencies of organophosphate hydrolysis and the large variability of PON1 levels observed in this and other populations, mothers and newborns homozygous for PON1Q192 with very low plasma levels of this alloform are predicted to be a subpopulation uniquely vulnerable to adverse effects from DZ/DZO exposures and especially CPS/CPO exposures. One particular concern would be exposure of a mother with very low PON1 status carrying a fetus that had not yet developed the capacity for self-protection against organophosphate exposure. PON1Q192 homozygous mothers with very low PON1 status are also predicted to be unable to pass on to their offspring a PON1 allele that would be protective against exposure. The father of course would contribute one PON1 allele to the child; however, it would need to be a high expressing allele and would take from 6 months to 2 years following birth to be fully protective. Although the usual precautions need to be observed in extrapolating data generated in an animal model system

to predict outcomes in human exposures, genetically manipulating a single gene in an inbred strain of mice is nonetheless informative. PON1 appears to be the only major enzyme in the plasma of both mice and humans that hydrolyzes chlorpyrifos oxon and diazoxon, as can be seen from the colinearity of the plots of rates of hydrolysis of one substrate against others in human populations [28] and from the dramatic consequences of deleting this gene in mice [34,46]. Replacing the mouse PON1 gene with each of the human PON1192 alleles provides a better means of extrapolating between the two species, even though other pathways may contribute somewhat to the detoxication. Even among humans, there is variability in levels and efficiencies of other contributory detoxication enzymes, such as carboxylesterases and cytochromes P450. Several factors are likely to contribute to the risk of adverse health effects due to exposures to CPS/CPO and DZ/DZO: (i) the level of exposure; (ii) the percentage of oxon residue or other toxic derivatives in the exposure; (iii) the level of enzyme (measured by AREase assay); (iv) the catalytic efficiency of an individuals PON1 to detoxify organophosphate metabolites (determined by the Q192R polymorphism); and (v) the as yet uncharacterized genetic variability of the cytochromes P450, carboxylesterases and other enzymes that participate significantly in the detoxication of these organophosphorus compounds. Additional research is needed to determine the relative contributions of PON1, cis- and trans-acting factors that modulate PON1 levels, cytochromes P450 and other enzymes, as well as the genetic and environmental factors that modulate the levels of enzymes involved in detoxication of parent compounds and their respective oxons. A recently reported method of haplotyping using emulsion PCR should be useful in understanding cis factors involved in regulation of protein levels [64]. Research is also needed to determine the exposure levels of specific residues (trichlorophenol, CPS, CPO, diethylphosphate and other toxic metabolites) required for modelling [48,65] the effect of PON1 status on the consequences of exposures. Evaluation of PON1 status is not only important for determining risk of organophosphate exposure, but also for understanding the role of PON1 in modulating other risks associated with the variability of normal physiological functions of PON1, as well as the role of PON1 in the metabolism of other xenobiotics, including drugs [66]. Multiple results indicating a prominent role for PON1 status in risk for vascular disease have been published previously [40,46,67,68]. More recently, PON1 has been shown to inactivate the quorum sensing signal secreted by Pseudomonads [66,69]; however, data demonstrating the
PON1 status and predicted organophosphate sensitivity Furlong et al. 189
in-vivo importance of this activity have not yet been reported. In summary, the range of variability of PON1 status observed in this study, taken together with data from humanized mice expressing hPON1Q192 or hPON1R192 in place of mouse PON1, predict a 65-fold variability in DZO sensitivity and a 131164-fold range in sensitivity to CPO exposure in this population, with an average fourfold difference in sensitivity to DZO exposure and an average eight-to ten-fold variability in sensitivity to CPO between groups of mothers and their newborns. These data predict that most, if not all, newborns, as well as a subpopulation of adults, will exhibit significantly increased sensitivity to organophosphate exposure. These findings highlight the significance of understanding the susceptibility of young children to organophosphate exposure and developing science-based risk standards for pesticide regulation as required by the 1996 Food Protection Act.

Acknowledgements

We gratefully acknowledge CHAMACOS staff, students and community partners, and especially the CHAMACOS participants and their families, without whom this study would not have been possible. We thank Katie Kogut and Erin Weltzien for their help with statistical analyses and Dr Toby Cole for helpful comments and suggestions.

References

Barr D, Bravo R, Weerasekera G, Caltabiano L, Whitehead R. Concentrations of dialkyl phosphate metabolites of organophosphorus pesticides in the US population. Environ Health Perspect 2004; 112: 186200. 2 Hill RH Jr, Head SL, Baker S, Gregg M, Shealy DB, Bailey SL, et al. Pesticide residues in urine of adults living in the United States: reference range concentrations. Environ Res 1995; 71:99108. 3 Loewenherz C, Fenske RA, Simcox NJ, Bellamy G, Kalman D. Biological monitoring of organophosphorus pesticide exposure among children of agricultural workers in central Washington State. Environ Health Perspect 1997; 105:344353. 4 Simcox NJ, Camp J, Kalman D, Stebbins A, Bellamy G, Lee IC, Fenske R. Farmworker exposure to organophosphorus pesticide residues during apple thinning in central Washington State. Am Ind Hyg Assoc J 1999; 60: 752761. 5 Berkowitz GS, Obel J, Deych E, Lapinski R, Godbold J, Liu Z, et al. Exposure to indoor pesticides during pregnancy in a multiethnic, urban cohort. Environ Health Perspect 2003; 111:7984. 6 Whyatt RM, Barr DB, Camann DE, Kinney PL, Barr JR, Andrews HF, et al. Contemporary-use pesticides in personal air samples during pregnancy and blood samples at delivery among urban minority mothers and newborns. Environ Health Perspect 2003; 111:749756. 7 Eskenazi B, Bradman A, Gladstone EA, Jaramillo S, Birch K, Holland NT. CHAMACOS: a longitudinal birth cohort study: lessons from the fields. J Child Health 2003; 1:327. 8 Bradman A, Eskenazi B, Barr DB, Bravo R, Castorina R, Chevrier J, et al. Organophosphate urinary metabolite levels during pregnancy and after delivery in women living in an agricultural community. Environ Health Perspect 2005; doi: 101289/ehp.7894 [Epub ahead of print]. 9 Bradman A, Barr DB, Claus Henn BG, Drumheller T, Curry C, Eskenazi B. Measurement of pesticides and other toxicants in amniotic fluid as a potential biomarker of prenatal exposure: a validation study. Environ Health Perspect 2003; 111:17821789. 10 Castorina R, Bradman A, McKone TE, Barr DB, Harnly ME, Eskenazi B. Cumulative organophosphate pesticide exposure and risk assessment among pregnant women living in an agricultural community: a case study 26

from the chamacos cohort. Environ Health Perspect 2003; 111: 16421648. Fenske RA, Lu C, Barr D, Needham L. Childrens exposure to chlorpyrifos and parathion in an agricultural community in central Washington State. Environ Health Perspect 2002; 110:549553. USEPA 2000. Chlorpyrifos revised risk assessment and agreement with registrants. http://www.epa.gov/pesticides/op/chlorpyrifos/agreement.pdf USEPA 2001. Diazinon revised risk assessment and agreement with registrants. http://www.epa.gov/pesticides/op/diazinon/agreement.pdf Sherman JD. Organophosphate pesticides neurological and respiratory toxicity. Toxicol Ind Health 1995; 11:3339. Eskenazi B, Bradman A, Castorina R. Exposures of children to organophosphate pesticides and their potential adverse health effects. Environ Health Perspect 1999; 107 (Suppl 3):409419. Young JG, Eskenazi B, Gladstone EA, Bradman A, Pedersen L, Johnson C, et al. Association between in utero organophosphate pesticide exposure and abnormal reflexes in neonates. Neurotoxicology 2005; 26:199209. Garcia SJ, Seidler FJ, Slotkin TA. Developmental neurotoxicity of chlorpyrifos: targeting glial cells. Env Toxicol Pharmacol 2005; 19: 455461. Moser VC, Padilla S. Age-and gender-related differences in the time-course of behavioral and biochemical effects produced by oral chlorpyrifos in rats. Toxicol Appl Pharmacol 1998; 149:107119. Sheets L. A consideration of age-dependent differences in susceptibility to organophosphorus and pyrethroid insecticides. Neurotoxicology 2000; 21:5763. Padilla S, Buzzard J, Moser VC. Comparison of the role of esterases in the differential age-related sensitivity to chlorpyrifos and methamidophos. Neurotoxicology 2000; 21:4956. Zheng Q, Olivier K, Won YK, Pope CN. Comparative cholinergic neurotoxicity of oral chlorpyrifos exposures in preweanling and adult rats. Toxicol Sci 2000; 55:124132. Pope CN, Chakraborti TK, Chapman ML, Farrar JD, Arthun D. Comparison of in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorothioate insecticides. Toxicology 1991; 68:5161. Chen J, Kumar M, Chan W, Berkowitz G, Wetmur JG. Increased influence of genetic variation on PON1 activity in neonates. Environ Health Perspect 2003; 111:14031409. Cole T, Jampsa RL, Walter BJ, Arndt TL, Richter RJ, Shih DM, et al. Expression of human paraoxonase (PON1) during development. Pharmacogenetics 2003; 13:357364. Ecobichon DJ, Stephens DS. Perinatal development of human blood esterases. Clin Pharmacol Ther 1973; 14:4147. Mueller RF, Hornung S, Furlong CE, Anderson J, Giblett ER, Motulsky AG. Plasma paraoxonase polymorphism: a new enzyme assay, population, family, biochemical, and linkage studies. Am J Hum Genet 1983; 35:393408. Augustinsson K, Barr M. Age variation in plasma arylesterase activity in children. Clin Chim Acta 1963; 8:568573. Davies HG, Richter RJ, Keifer M, Broomfield CA, Sowalla J, Furlong CE. The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin. Nat Genet 1996; 14:334336. Furlong CE, Costa LG, Hassett C, Richter RJ, Adler DA, Disteche CM, et al. Human and rabbit paraoxonases: purification, cloning, sequencing, mapping and role of polymorphism in organophosphate detoxification. Chem Biol Interact 1993; 87:3548. Hassett C, Richter RJ, Humbert R, Chapline C, Crabb JW, Omiecinski CJ, Furlong CE. Characterization of cDNA clones encoding rabbit and human serum paraoxonase: the mature protein retains its signal sequence. Biochemistry 1991; 30:1014110149. Sorenson RC, Bisgaier CL, Aviram M, Hsu C, Billecke S, La Du BN. Human serum paraoxonase/arylesterases retained hydrophobic N-terminal leader sequence associates with HDLs by binding phospholipids: apolipoprotein A-I stabilizes activity. Arterioscler Thromb Vasc Biol 1999; 19:22142225. Humbert R, Adler DA, Disteche CM, Hassett C, Omiecinski CJ, Furlong CE. The molecular basis of the human serum paraoxonase activity polymorphism. Nat Genet 1993; 3:7376. Adkins S, Gan KN, Mody M, La Du BN. Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes. Am J Hum Genet 1993; 52:598608. Li WF, Costa LG, Richter RJ, Hagen T, Shih DM, Tward A, et al. Catalytic efficiency determines the in-vivo efficacy of PON1 for detoxifying organophosphorus compounds. Pharmacogenetics 2000; 10:767779. Brophy VH, Jampsa RL, Clendenning JB, McKinstry LA, Jarvik GP, Furlong CE. Effects of 50 regulatory region polymorphisms on paraoxonase (PON1) expression. Am J Hum Genet 2001; 68:14281436.

Deakin S, Leviev I, Brulhart-Meynet M-C, James RW. Paraoxonase-1 promoter haplotypes and serum paraoxonase: a predominant role for polymorphic position 107, implicating the Sp1 transcription factor. Biochem J 2003; 372:643649. Suehiro T, Nakamura T, Inoue M, Shiinoki T, Ikeda Y, Kumon Y, et al. A polymorphism upstream from the human paraoxonase (PON1) gene and its association with PON1 expression. Atherosclerosis 2000; 150:295298. Li WF, Costa LG, Furlong CE. Serum paraoxonase status: a major factor in determining resistance to organophosphates. J Toxicol Environ Health 1993; 40:337346. Richter RJ, Furlong CE. Determination of paraoxonase (PON1) status requires more than genotyping. Pharmacogenetics 1999; 9:745753. Jarvik GP, Rozek LS, Brophy VH, Hatsukami TS, Richter RJ, Schellenberg GD, Furlong CE. Paraoxonase (PON1) phenotype is a better predictor of vascular disease than is PON1 (192) or PON1 (55) genotype. Arterioscler Thromb Vasc Biol 2000; 20:24412447. Jarvik GP, Jampsa R, Richter RJ, Carlson CS, Rieder MJ, Nickerson DA, Furlong CE. Novel paraoxonase (PON1) nonsense and missense mutations predicted by functional genomic assay of PON1 status. Pharmacogenetics 2003; 13:291295. Leviev I, Deakin S, James RW. Decreased stability of the M54 isoform of paraoxonase as a contributory factor to variations in human serum paraoxonase concentrations. J Lipid Res 2001; 42:528535. Main AR. The role of A-esterase in the acute toxicity of paraoxon, TEPP and parathion. Can J Biochem Physiol 1956; 34:197216. Costa LG, McDonald BE, Murphy SD, Omenn GS, Richter RJ, Motulsky AG, Furlong CE. Serum paraoxonase and its influence on paraoxon and chlorpyrifos-oxon toxicity in rats. Toxicol Appl Pharmacol 1990; 103: 6676. Furlong CE, Richter RJ, Seidel SL, Costa LG, Motulsky AG. Spectrophotometric assays for the enzymatic hydrolysis of the active metabolites of chlorpyrifos and parathion by plasma paraoxonase/ arylesterase. Anal Biochem 1989; 180:242247. Shih DM, Gu L, Xia YR, Navab M, Li WF, Hama S, et al. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature 1998; 394:284287. Li WF, Furlong CE, Costa LG. Paraoxonase protects against chlorpyrifos toxicity in mice. Toxicol Lett 1995; 76:219226. Cole T, Walter B, Shih D, Tward A, Lusis AJ, Timchalk C, et al. Toxicity of chlorpyriphos oxon in a transgenic mouse model of the human paraoxonase (PON1) Q192R polymorphism. Pharmacogenet Genom 2005; 15: 589598. Brophy VH, Jarvik GP, Furlong C. PON1 polymorphisms. In: Costa LG, Furlong C, editors. Paraoxonase (PON1) in health and disease: basic and clinical aspects. Boston, Massachusetts: Kluwer Academic Press; 2002. pp. 5377. DPR. Pesticide Use Reporting 2001 Summary Data: Department of Pesticide Regulation. Sacramento, California: California Environmental Protection Agency; 2002. Eskenazi B, Harley K, Bradman A, Weltzien E, Jewell NP, Barr DB, et al. Association of in utero organophosphate pesticide exposure and fetal growth and length of gestation in an agricultural population. Environ Health Perspect 2004; 112:11161124.

Richter R, Jampsa R, Jarvik GP, Costa LG, Furlong CE. Determination of paraoxonase 1 (PON1) status and genotypes at specific polymorphic sites. Curr Protoc Toxicol 2004; 4.12.14.12.19. Blatter-Garin M-C, Abbot C, Messmer S, Mackness MI, Durrington P, Pometta D, James RW. Quantification of human serum paraoxonase by enzyme-linked immunoassay: population differences in protein concentrations. Biochem J 1994; 304:549554. StataCorp. Stata Statistical Software: release 8.0. College Station, Texas: Stata Corporation; 2003. Berkowitz GS, Wetmur JG, Birman-Deych E, Obel J, Lapinski RH, Godbold JH, et al. In-utero exposure, maternal paraoxonase activity and head circumference. Environ Health Perspect 2004; 112:338391. Burlina A, Michielin E, Galzinga L. Characteristics and behaviour of arylesterase in human serum and liver. Eur J Clin Invest 1977; 7:1720. Yuknavage KL, Fenske RA, Kalman DA, Keifer MC, Furlong CE. Simulated dermal contamination with capillary samples and field cholinesterase biomonitoring. J Toxicol Env Health 1997; 51:3555. California EPA 2002. Pesticide use reporting 2001 Summary data. http://www.cdpr.ca.gov/docs/pur/pur01rep/01_pur.htm Huff R, Abou-Donia MB. In vitro effect of chlorpyrifos oxon on muscarinic receptors and adenylate cyclase. Neurotoxicology 1995; 16:281290. Li W-F. Dissertation: development of a mouse model for studying paraoxonase. Seattle, Washington: University of Washington, School of Public Health and Community Medicine, Department of Environmental Health; 1993. Li W-F, Matthews C, Disteche CM, Costa LG, Furlong CE. Paraoxonase (Pon1) gene in mice: sequencing, chromosomal location and developmental expression. Pharmacogenetics 1997; 7:137144. Moser VC, Chanda SM, Mortensen SR, Padilla S. Age- and gender-related differences in sensitivity to chlorpyrifos in the rat reflect developmental profiles of esterase activities. Toxicol Sci 1998; 46:211222. Hosokawa M, Endo T, Fujisawa M, Hara S, Iwata N, Sato Y, Satoh T. Interindividual variation in carboxylesterase levels in human liver microsomes. Drug Metabol Dispos 1995; 23:10221027. Wetmur JG, Kumar M, Zhang L, Palomeque C, Wallenstein S, Chen J. Molecular haplotyping by linking emulsion PCE: analysis of paraoxonase 1 haplotypes and phenotypes. Nucleic Acids Res 2005; 33:26152619. Timchalk C, Poet TS, Hinman MN, Busby AL, Kousba AA. Pharmacokinetic and pharmacodynamic interaction for a binary mixture of chlorpyrifos and diazinon in the rat. Toxicol Appl Pharmacol 2005; 205:3142. Draganov DI, Teiber JF, Speelman A, Osawa Y, Sunahara R, La Du BN. Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities. J Lipid Res 2005; 46:12391247. Mackness B, Davies GK, Turkie W, Lee E, Roberts DH, Hill E, et al. Paraoxonase status in coronary heart disease: are activity and concentration more important than genotype? Atherscler Thromb Vasc Biol 2001; 21:14511457. Mackness M, Mackness B. Paraoxonase 1 and atherosclerosis: is the gene or the protein more important? Free Rad Biol Med 2004; 37:13171323. Ozer EA, Pezzulo A, Shih DM, Chun C, Furlong C, Lusis AJ, et al. Human and murine paraoxonase 1 are host modulators of P. aeruginosa quorumsensing. FEMS Microbiol Lett 2005; in press.