Longitudinal changes in PON1 enzymatic activities in Mexican–American mothers and children with different genotypes and haplotypes

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Abstract

The paraoxonase 1 (PON1) enzyme prevents low-density lipoprotein oxidation and also detoxifies the oxon derivatives of certain neurotoxic organophosphate (OP) pesticides. PON1 activity in infants is low compared to adults, rendering them with lower metabolic and antioxidant capacities. We made a longitudinal comparison of the role of genetic variability on control of PON1 phenotypes in Mexican–American mothers and their children at the time of delivery (n = 388 and 338, respectively) and again 7 years later (n = 280 and 281, respectively) using generalized estimating equations models. At age 7, children's mean PON1 activities were still lower than those of mothers. This difference was larger in children with genotypes associated with low PON1 activities (PON1108TT, PON1192QQ, and PON1909CC). In mothers, PON1 activities were elevated at delivery and during pregnancy compared to 7 years later when they were not pregnant (p < 0.001). In non-pregnant mothers, PON1 polymorphisms and haplotypes accounted for almost 2-fold more variation of arylesterase (AREase) and chlorpyrifos-oxonase (CPOase) activity than in mothers at delivery. In both mothers and children, the five PON1 polymorphisms (192, 55, −108, −909, −162) explained a noticeably larger proportion of variance of paraoxonase activity (62–78%) than AREase activity (12.3–26.6%). Genetic control of PON1 enzymatic activity varies in children compared to adults and is also affected by pregnancy status. In addition to known PON1 polymorphisms, unidentified environmental, genetic, or epigenetic factors may also influence variability of PON1 expression and therefore susceptibility to OPs and oxidative stress.

Introduction

Paraoxonase 1 (PON1) is a high-density-lipoprotein (HDL)-associated enzyme that plays a role in both organophosphate (OP) sensitivity and oxidative stress (Azarsiz et al., 2003). PON1 can metabolize the toxic oxon derivatives of several OP pesticides, which are known to be acutely neurotoxic (Costa et al., 2005a). There is growing evidence that PON1 may play a role in diseases related to oxidative stress including diabetes and heart disease (Li et al., 2003, Li et al., 2005, Bhattacharyya et al., 2008). In vitro and in vivo studies have demonstrated that PON1 has antioxidant properties, preventing LDL and HDL oxidation (Aviram and Rosenblat, 2004) and protecting against atherosclerosis (Tward et al., 2002, Rosenblat et al., 2006). Current studies suggest lipophilic lactones are the primary substrate for PON1 (Draganov et al., 2005, Khersonsky and Tawfik, 2005), and it is through this mechanism that PON1 is involved in lipid peroxidation. Although PON1 was named for its esterase activity towards OPs, the endogenous function of this enzyme is more likely its lipolactonase activity (Draganov et al., 2005). In humans, there is a wide variability of PON1 enzymatic activities among adults (Deakin and James, 2004). Individuals with low PON1 activity may be more susceptible to pesticide exposures and oxidative stress since their metabolic capacity and antioxidant defenses are lower compared to those with average or high PON1 activities. Thus, understanding the determinants of PON1 variability, including genetics and age, and how they confer susceptibility to disease or exposures may have broad public health significance.

Several common polymorphisms in the coding and promoter regions of the PON1 gene influence substrate-specific PON1 enzyme activities (Ferre et al., 2003, Costa et al., 2005b). The single nucleotide polymorphism (SNP) at codon 192 leads to an amino acid substitution from the active-site residue glutamine (Q) to arginine (R) and the catalytic efficiency of the PON1192 R alloform towards the oxon derivatives of OP pesticides parathion and chlorpyrifos is greater than that of the PON1192 Q alloform in in vitro substrate-specific assays. Animal experiments in transgenic mice expressing human PON1192 Q and R alloforms have demonstrated that indeed mice expressing the R alloform are more resistant to chlorpyrifos-oxon (CPO) exposure than mice expressing the Q alloform (Cole 2005). For paraoxon, however, the catalytic efficiency even in the faster R alloform is too slow to provide any protection from in vivo exposures (Li et al., 2000). Recent studies found that the PON1192 genotype explains a large portion of the variability of in vitro PON1 activity towards paraoxon (POase activity); it accounts for 59% of the variability among Caucasian and African–American adults (Bhattacharyya et al., 2008) and 48% of the variability in a Mexican–American population (Rainwater et al., 2009). Several promoter polymorphisms are also known to influence PON1 expression including PON1108, PON1162, and PON1909 (positions are relative to the translation start site). The PON1108 SNP exerts the most noticeable effect on PON1 quantity, as measured by arylesterase activity, accounting for 22.4% of the variability. The PON1108CC genotype is associated with 2-fold higher PON1 levels compared to the PON1108TT genotype (Brophy et al., 2001, Deakin et al., 2003). The association of the SNPs at positions −162 and −909 with AREase activity likely is due in part to their strong linkage disequilibrium (LD) with the PON1108 SNP (Brophy et al., 2001, Holland et al., 2006). Similarly, the coding SNP PON1L55M is also associated with AREase activity; however, most of this effect is attributable to LD with PON1108 (Brophy et al., 2001). While several studies have described the important genetic contribution of these PON1 SNPs on phenotypic variation in multiple populations, few have characterized how the relative influence of genetic control may change through different stages of childhood and by pregnancy status. Furthermore, although genetic polymorphisms account for a large portion of PON1 variability, it is not sufficient in epidemiological studies to consider PON1 genotypes alone (Richter and Furlong, 1999). PON1 phenotypes range broadly even between individuals with the same PON1 genotypes because enzyme quantity also varies within these groups (Furlong et al., 2006, Holland et al., 2006). Therefore, studies that measure PON1 activities are more informative than studies that rely solely on PON1 genotype data.

Children are particularly vulnerable to environmental exposures because they practice behaviors that can lead to increased exposure and often have lower metabolic capacities than adults (Landrigan et al., 2004, Neri et al., 2006, Wigle et al., 2007). For example, children's susceptibility to the toxic metabolites of OPs and oxidative stress may be heightened as several studies have demonstrated that PON1 activity is lower in newborns compared to adults (Chen et al., 2003, Holland et al., 2006). Early hypotheses suggested that PON1 developmental expression reaches mature levels at or near age 2 (Cole et al., 2003); however, we recently followed a large cohort of Mexican–American children from birth to age 7 and found that their PON1 activities continue to increase past age 2 until at least age 7 (Huen et al., 2009a). This age-dependent increase of PON1 enzymatic activity was modified by genetic polymorphisms. For example, children with PON1192 R alleles and PON1108 C alleles experienced a steeper rise in activity as they got older compared to children with PON1192 Q alleles and PON1108 T alleles. These findings suggest that the window of susceptibility to both oxidative stress and OP exposure may be much longer than previously believed and children with certain genotypes may be particularly vulnerable.

Initially, we reported PON1 activity in a subset of 130 mother–child pairs from the Center for Health Assessment in Children and Mothers of Salinas Valley (CHAMACOS) cohort and determined the effects of genotypes and haplotypes on PON1 phenotype and status (Furlong et al., 2006, Holland et al., 2006). In the present study, we performed a longitudinal comparison of the role of genetic control on PON1 enzymatic activities in the entire CHAMACOS birth cohort of mothers and their children at the time of birth and also 7 years later. We also compared PON1 activities between mothers and children at both time points and determined differences in PON1 activities in mothers during pregnancy, at delivery, and 7 years later when they were not pregnant.

Section snippets

Study subjects

CHAMACOS is a longitudinal birth cohort study of the effects of pesticide and other environmental exposures on neurodevelopment, growth, and respiratory disease in children from primarily Mexican–American families (Eskenazi et al., 2003). The Salinas Valley, which is located in Monterey County, CA, is intensively farmed with approximately 200,000 kg of OPs applied annually (DPR, 2007). Six hundred and one pregnant women were enrolled in 1999–2000, and 531 were followed through the birth of a

PON1 polymorphisms and haplotypes

Genotype distributions did not deviate significantly from Hardy–Weinberg equilibrium. Allelic frequencies in mothers and children for all five SNPs are presented in Table 1. For SNPs PON1909, PON1108, and PON1192 allele frequencies were approximately equal in this population. The frequencies of the major allele for PON1162 (G) and PON155 (L) were 81% and 82%, respectively. Allele and genotype frequencies did not differ significantly between mothers and children (χ2 test: p-value > 0.05 for

Discussion

In this study, three substrate-specific measures of PON1 quantity and activity were measured in over 400 mothers and children at the time of delivery, when the children were 7 years old, and also in mothers during pregnancy. Although children's PON1 activities increased about 3.5-fold between birth and age 7, they still remained 1.8–4.3% lower than levels measured in their non-pregnant mothers. Mothers had significantly higher levels of PON activity during pregnancy than 7 years later when they

Acknowledgments

We are grateful to the laboratory and clinical staff and participants of the CHAMACOS study for their contributions. We would also like to thank Dr. Kenneth Beckman for his assistance with PON1 genotyping and Dr. Clement Furlong and Rebecca Richter for their advice on PON1 enzymatic assays. This publication was made possible by grant numbers R826886 and R82670901 from the US Environmental Protection Agency (EPA) and R01ESO12503-03 and PO1 ES009605 from the National Institute of Environmental

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