Grantee Research Project Results

Final Report: Genetics of Phthalate and Bisphenol A Risk in Minority Populations (Individual Susceptibility)

EPA Grant Number: R831711C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R831711
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Center for Research on Early Childhood Exposure and Development in Puerto Rico
Center Director: Alshawabkeh, Akram
Title: Genetics of Phthalate and Bisphenol A Risk in Minority Populations (Individual Susceptibility)
Investigators: Wetmur, James G. , Wolff, Mary S. , Chen, Jia
Institution: Mount Sinai School of Medicine
EPA Project Officer: Callan, Richard
Project Period: November 1, 2003 through October 31, 2008 (Extended to October 31, 2010)
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2003) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health

Objective:

This progress report covers the period from 11/1/2003 to 10/31/2010. Discussion of the specific aims has been reversed to follow chronological order. The specific aims of this project were:

Specific Aims:

Determine HDL levels for the existing birth cohort and carry out a detailed genotype/phenotype reanalysis (a continuation of PON1 phenotype/genotype studies from the previous funding period). Genotype and haplotype new members of the extended birth cohort for PON1 polymorphisms. Collaborate with Project 2 to examine PON1 genotype and phenotype effects on developmental outcomes.
Genotype a common missense polymorphism in human UGT2B7 in our population of African-Americans, Caucasians and Hispanics and its association with urinary metabolites. Identify and assess common promoter, coding region and splice-site polymorphisms in human UGT2B7 in our African-Americans, Caucasians and Hispanics, establish the common haplotypes, and their association with exposure. Collaborate with Project 2 to examine human UGT2B7 genotype/haplotype effects on developmental outcomes.
Examine salivary lipase activity as a biomarker for conversion of phthalate diesters to phthalate monoesters. Determine expression levels of the three linked human lipase genes in human salivary glands. Determine racial/ethnic differences in the frequencies of the common polymorphisms in the salivary human lipase gene(s) and in salivary lipase activity. Examine salivary lipase activity in relation to urinary levels of phthalate metabolites. Examine salivary lipase polymorphisms and haplotypes in relation to urinary levels of phthalate metabolites. Obtain pilot data on the extent of glucuronidation of phthalate monoesters in urine. Collaborate with the epidemiology project (Project 2-R831711C002) to examine association of lipase genotype and phenotype with child developmental outcomes.

Summary/Accomplishments (Outputs/Outcomes):

Specific Aim 1 (a continuation of PON1 phenotype/genotype studies from the previous funding period): Determine HDL levels for the existing birth cohort and carry out a detailed genotype/phenotype reanalysis. There was a significant effect of PON1 genotype [P = 0.003 CC vs. TT or CC; P(trend) = 0.01] and BMI and possibly HDL, after adjusting for ethnicity, but not PON1 phenotype. There were no other significant interactions.

Genotype and haplotype new members of the extended birth cohort for PON1 polymorphisms. Collaborate with Project 2 (R831711C002) to examine PON1 genotype and phenotype effects on developmental outcomes. These studies have been completed and form part of the analyses in the publications summarized below (Berkowitz et al. 2004; Engel et al. 2007; Wolff et al. 2007). The Engel et al. publication is described Project 2 (R831711C002). The Wolff et al. publication extended the work of the Berkowitz et al. publication. In it, we measured biomarkers of maternal exposure to DDE, PCB, and OP metabolites in the third trimester of pregnancy among 404 mothers in a multiethnic cohort in New York City. We also determined maternal paraoxonase (PON1), butyrylcholinesterase (BuChe), and PON1Q192R gene variant. Higher multivariate-adjusted DDE levels (but not PCB) were associated with lower birth weight (-98 g/log₁₀ DDE, p = 0.096) and head circumference (- 0.54 cm/log₁₀ DDE, p = 0.030). DDE and PCB levels were not related to birth length, Ponderal index, or gestational age. Birth length was shorter for mothers with PON192RR slow genotype compared with PON192QQ (p = 0.026), and head circumference was inversely associated with maternal PON1 activity (p = 0.004). With slow-activity PON1 or PON192, urinary diethylphosphates (ΣDEPs) were associated with lower birth weight and dimethylphosphates (ΣDMPs) with shorter birth length. No associations were found between birth outcomes and BuChe. In summary, we found suggestive relationships between prenatal environmental biomarkers and birth outcomes in this population. Maternal susceptibility factors including PON1 and maternal weight contributed to the observed effects.

In addition, we used PON1 for the development of our emulsion haplotyping system and for analysis of the relative advantage of using measured versus inferred haplotypes, as described in the publications summarized below (Chen et al. 2005; Wallenstein et al. 2006; Wetmur and Chen 2008; Wetmur et al. 2005; Wetmur and Chen, 2011). The method is summarized in Wetmur et al. and Wetmur and Chen. Specifically, Linking emulsion PCR (LE-PCR) enables formation of minichromosomes preserving phase information of two polymorphic loci, hence the haplotype. Emulsion PCR confines two amplicons of two linked polymorphic sites on a single template molecule to one aqueous-phase droplet. Linking PCR uses biotinylated, overlapping linking primers to connect these amplicons in the droplet. After LE-PCR, unlinked amplicons are removed on streptavidin coated magnetic beads and single-stranded runoff products are capped by primer extension. Quantitative ASPCR can then be used to ascertain the haplotypes of the two polymorphic loci on the minichromosomes. Using LE-PCR, we determined the human paraoxonase-1 [PON1] molecular haplotypes at three loci (909g > c, L55M, Q192R ) in women who were compound heterozygotes for 909g>c/L55M (n = 89), 909g > c/Q192R (n = 77) and L55M/Q192R (n = 68). We observed a strong association between PON1 substrate specificity (paraoxon/phenylacetate substrate activity ratios) and 909g>c/Q192R haplotype. We have demonstrated here a powerful molecular haplotyping technology that can be applied in population studies. The Chen et al. and Wallenstein et al. papers describe application of the new methodology to the Children's Center birth cohort.

Self-reported race/ethnicity is frequently used in epidemiology studies to assess an individual's (participant's) background origin. However, in admixed populations such as Hispanic, self-reported race/ethnicity may not accurately represent them genetically because they are admixed of European, African and Native American. Thus, they can be self-identified as any race/ethnicity based on choices of the questionnaires. We utilized the resources of the birth cohort from Project 2 (R831711C002); we estimated the proportions of genetic admixture in an ethnically diverse population of 396 mothers and 188 children born to the mothers with 35 ancestry informative markers (AIMs). Mothers self-identified as Black and White possessed the ancestry proportions of 77.6% African and 75.1% European respectively; racial composition among self-identified Hispanics were 29.2% European, 26.0% African and 44.8% Native American. To demonstrate the utility of AIMs, we re-evaluated the PON1 genotype-phenotype associations that have been published previously (Chen et al. 2003). We showed improved fitness after incorporating AIMs into the models. Although limited improvement was observed in mothers, more profound improvement was observed in infants. In summary, a minimal set of 35 AIMs are sufficient to detect population substructure and estimate the proportion of individual genetic admixture. Our results, published in Lee et al. 2010, suggest that genetic structure in Hispanic population should be estimated by AIMs rather than relying on self-reported race and ethnicity.

Specific Aim 2: Genotype a common missense polymorphism in human UGT2B7 in our population of African-Americans, Caucasians and Hispanics and its association with urinary metabolites. Identify and assess common promoter, coding region and splice-site polymorphisms in human UGT2B7 in our African-Americans, Caucasians and Hispanics, establish the common haplotypes, and their association with exposure. Collaborate with Project 2 (R831711C002) to examine human UGT2B7 genotype/haplotype effects on developmental outcomes.

We found that determination of the common H268Y polymorphism was sufficient. We extended this bisphenol A aim to study nine phenols. The exposure data have been published (Wolff et al. 2008). The phenol association analyses (manuscript in preparation) were limited to five of nine phenols with > 60% detectable values (> 79-100% detectability; the medians were 1.3-53 µg/L).

The reported phenol metabolite values are based on both free and glucuronidated forms. Of the five phenols with detectable levels, TCS had the highest free concentration at 7%, with no significant trend based on genotype. Thus, total concentration and bound (conjugated) concentrations are nearly equal. The same significant associations were seen in a multivariate model adjusted for creatinine, maternal age and race. The association of phenol biomarkers with UGT2B7 phenotypes found no significant associations, although both BPA and TCS, phenols that need no phase I enzyme, were trended lower for individuals carrying the Y allele even after correcting for race/ethnicity. No such trend was seen for 24DCP or 25DCP, both of which are formed from parent compounds that must be oxidized to phenols by cytochrome P450s. Although no association was seen with BP3, which does not require a phase I enzyme, it is an ingredient of sunscreen and the Y form of UGT2B7 is more prevalent in Whites than Blacks.

Specific Aim 3: Examine salivary lipase activity as a biomarker for conversion of phthalate diesters to phthalate monoesters. Determine expression levels of the three linked human lipase genes in human salivary glands. Determine racial/ethnic differences in the frequencies of the common polymorphisms in the salivary human lipase gene(s) and in salivary lipase activity. Examine salivary lipase activity in relation to urinary levels of phthalate metabolites. Examine salivary lipase polymorphisms and haplotypes in relation to urinary levels of phthalate metabolites. Obtain pilot data on the extent of glucuronidation of phthalate monoesters in urine. Collaborate with the epidemiology project (Project 2-R831711C002)) to examine association of lipase genotype and phenotype with child developmental outcomes.

We examined six phthalates. The exposure data have been published (Wolff et al. 2008). The phthalate association studies examined a panel of phthalate biomarkers (manuscript in preparation).

For most analyses, the molecular sum of urinary MEP, MMP and MBP metabolites was combined as loMW and the molecular sum of DEHP, MBZP and MCPP metabolites as HMWP. The LMWP values were normalized to creatinine. The reported values are based on both free and glucuronidated metabolites. The majority of the metabolites were glucuronidated, with unbound monoesters representing 15, 15, 18, 20, 21, 23, 34, 36 and 44% for mEOHP, mEHHP, mBP, mEHP, mBzP, miBP, mECPP, mCPP and mEP, respectively. We determined the expression of lipase genes in human salivary gland and found only pancreatic PNLIP and gastric LIPF to be expressed, and both to be expressed at low levels. We measured lipase and amylase (for normalization) levels in saliva samples and found no association between salivary lipase activity and urinary phthalate metabolites. Based on analysis below that showed PNLIP and LIPF genotypes and haplotypes were associated with urinary phthalate metabolites, we conclude that the high lipase activities in the GI tract outweigh low level salivary lipase activities.

Associations between lipase genotypes and biomarkers were evaluated by a two-level model where the heterozygote and rare homozygote contributions were combined. All analyses were adjusted for race/ethnicity and creatinine. We observed strong association with HMWP and LMWP that differed by genotype. Although the SNP associations for the lipase genes were found after adjustment for race/ethnicity, to reduce the possibility of confounding by population stratification, we examined the haplotype structure and associations for the two lipase genes. Association analysis by haplotype adjusted for creatinine (restricted to >20 mg/dl) revealed strong associations.

Conclusions:

All publications for this project are reported under the Final Report for the Center (see Final Report for Grant No. R831711)

Journal Articles:

No journal articles submitted with this report: View all 93 publications for this subproject

Supplemental Keywords:

Biochemistry, Chemicals, childhood development, children's environmental health, Children's Health, children's vulnerablity, endocrine disrupting chemicals, endocrine disruptors, Environmental Chemistry, Environmental Exposure & Risk, environmental health, environmental management, exposure pathways, exposure studies, fast food, Health, Health Risk Assessment, human health, lipase, neurodevelopment, neurodevelopmental toxicity, obesity, paraoxonase, pesticide exposure, pesticides, phthalates, pollutants/toxics, RFA, Risk Assessment, Scientific Discipline, RFA, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, POLLUTANTS/TOXICS, Health Risk Assessment, Environmental Chemistry, Endocrine Disruptors - Human Health, Children's Health, endocrine disruptors, Biochemistry, Endocrine Disruptors - Environmental Exposure & Risk, Chemicals, Risk Assessment, Human Health Risk Assessment, neurodevelopmental toxicity, pesticides, pesticide exposure, phtalates, exposure studies, genetic polymorphisms, endocrine disrupting chemicals, children's vulnerablity, environmental health, children's environmental health, exposure pathways, childhood development

Relevant Websites:

http://www.mountsinai.org/patient-care/service-areas/children/areas-of-care/childrens-environmental-health-center/ Exit

Progress and Final Reports:

Original Abstract

Main Center Abstract and Reports:

R831711 Center for Research on Early Childhood Exposure and Development in Puerto Rico

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R831711C001 Growing Up Healthy in East Harlem (Community-Based Participatory Research)
R831711C002 Pesticides, Endocrine Disruptors, Childhood Growth and Development (Birth Cohort)
R831711C003 Genetics of Phthalate and Bisphenol A Risk in Minority Populations (Individual Susceptibility)

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