2015 Progress Report: Genetic Susceptibility

EPA Grant Number: R834514C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R834514
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: University of Washington Center for Child Environmental Health Risks Research (2010)
Center Director: Faustman, Elaine
Title: Genetic Susceptibility
Investigators: Faustman, Elaine , Furlong, Clement
Current Investigators: Furlong, Clement
Institution: University of Washington
EPA Project Officer: Callan, Richard
Project Period: September 25, 2009 through September 24, 2015 (Extended to September 24, 2016)
Project Period Covered by this Report: August 25, 2014 through September 24,2015
RFA: Children's Environmental Health and Disease Prevention Research Centers (with NIEHS) (2009) RFA Text |  Recipients Lists
Research Category: Children's Health , Health

Objective:

Since 1998, researchers of the University of Washington Center for Child Environmental Health Risks Research (the Center) have been using a multi-disciplinary research approach working in the lab, in the field, and in the community to understand the mechanisms that define children’s susceptibility to pesticides, identify the implications of this susceptibility for development and learning, and partner with our communities to translate our findings into risk communication, risk management, and prevention strategies.

The specific objectives of the laboratory-based research projects—a molecular mechanisms research project and a genetic susceptibility research project—are:

  1. To identify cellular, biochemical and molecular mechanisms that cause adverse developmental neurotoxicity of pesticides; and
  2. To identify susceptibility factors for developmental neurotoxicity of pesticides.

The overall goal of the Genetic Susceptibility Research Project is to develop specific biomarkers of exposure to organophosphate (OP) compounds, and to use these biomarkers to explore gene-environment interactions related to genetic variability in the paraoxonase (PON1) gene, particularly with respect to OP exposures that occur during early development.

Progress Summary:

Specific Aim 1 was to develop and validate immunomagnetic bead (IMB) isolation protocols for the biomarker proteins of interest: butyrylcholinesterase (BChE), acyl peptide hydrolase (APH), and acetylcholinesterase (AChE), and to use a proteomic approach to validate the use of these proteins as biomarkers of OP exposure.

We had demonstrated that a single step IMB protocol will purify BChE from plasma and APH from red cell extracts. The OP adducts to these proteins have been identified. The serine adduct from the active metabolite of tri-cresyl phosphates (TCP) was found to undergo a unique aging reaction. In vitro inhibition of BChE with the active metabolite of TCP, cyclic saligenin cresyl phosphate (CBDP) generated both a cresyl phosphoserine and an aged phosphoserine. However, in samples from exposed individuals, only the fully aged phosphoserine modification was observed. More recent experiments have shown that AChE does not age in the same way and retains a cresyl group. We had anti-AChE antibodies generated in chicken (Aves Labs, Tigard, OR) based on the sequence differences between human and chicken upstream from the active site serine. We have used these antibodies to develop a protocol for isolation of the human active site tryptic peptide using the chicken antibodies similar to the protocols that we have developed for the rapid isolation of BChE and APH. We currently are using this protocol to examine AChE adduction by OP insecticides as well as the neurotoxins to which children are exposed during flights in commercial aircraft, the triaryl phosphates.

A second biomarker protein, acylpeptide hydrolase (APH) has proven in our mouse in vivo experiments to be very sensitive to chlorpyrifos oxon (CPO) exposure and is useful because it has a half-life three-times as long as BChE (33 d vs. 11 d). We have prepared and sent to the Centers for Disease Control and Prevention (CDC) OP modified and unmodified APH as well as a small quantity of 15N-labeled APH produced in our E. coli expression system to serve as an internal standard when quantifying OP exposures.

The immunomagnetic bead/mass spec protocols are sufficiently sensitive to determine exposures from dried blood spots, which are much easier to collect, ship, and archive.

We also added a fourth potentially useful biomarker to our targeted proteins that are modified by OP exposures. While it has been known for many years, there is no carboxylesterase 1 (CES1) in human serum/plasma, nonetheless, we can pull sufficient CES1 out of white cells to analyze for OP adduction; in fact, CES1 also is known as monocyte carboxylesterase. It has the unique property of being modified by non-bioactivated triaryl phosphates.

Specific Aims 2 and 3 made use of knockout and humanized mouse models to evaluate interactions among biomarkers of OP sensitivity, exposure, and response during critical stages of development. The experiments in Aim 2 involved chronic exposure of pregnant mice to chlorpyrifos (CP) and CPO. The experiments in Aim 3 involve neonatal exposures.

For the experiments characterizing PON1 status as a biomarker of sensitivity to CP and CPO during gestation, the initial chronic dose-response studies in pregnant and nonpregnant mice demonstrated steep dose-response curves for inhibition of BChE and AChE, with higher doses leading to weight loss and fetal abnormalities. PON1-/- mice were more sensitive than PON1+/+mice to the effects of chronic CPO exposure. Fetuses were more resistant than dams to CPO inhibition of BChE, and tgHuPON1R192 females were more resistant than females expressing tgHuPON1Q192. In our earlier studies, dose-response experiments were completed for CPO that involved gestational exposure of mice of all four genotypes (n = 10 pregnant dams per treatment group). After timed matings were performed (n = 20 females per treatment group), mice with copulatory plugs were exposed transdermally to CPO daily from GD6 to GD17. PON1+/+, tgHuPON1Q192, and tgHuPON1R192 mice were exposed to vehicle (acetone) or to CPO at 0.75 mg/kg/d or 0.85 mg/kg/d. PON1-/- mice were exposed to vehicle alone, or to CPO at 0.50 mg/kg/d or 0.75 mg/kg/d. Dams were sacrificed at GD18, and fetal and maternal tissues were dissected for measurement of enzyme inhibition in different tissues. Additionally, fetal brains were placed in RNA later and analyzed for gene expression using microarrays. At GD18 there was greater inhibition of BChE in the plasma of fetuses from PON1-/- and tgHuPON1Q192 dams, as compared to the plasma of fetuses from PON1+/+ and tgHuPON1R192 dams. Thus, the PON1 status of the dam clearly influenced CPO toxicity to the fetus. The gene expression analysis showed that gene expression in brains of the fetuses of the PON1-/- mice was more significantly affected than in brains of fetuses of wild type mice. Also, gene expression in the brains of fetuses of the PON1R192 mice was less affected than expression in the brains of fetuses of PON1Q192 mice, consistent with the higher efficiency of hydrolysis of CPO by the PON1R192 alloform. Thus, PON1 status was important in modulating CPO exposure both pre- and postnatally.

Also interesting was the observation that RBC [red blood cell] APH showed a better correlation with brain AChE inhibition than the classic biomarker protein BChE, validating our use of APH as an important and useful biomarker protein for OP exposures. Similar results in fetuses were observed when the four mouse strains were exposed to diazoxon (DZO) during gestation.

Significance

These studies have provided information on the consequences of OP exposure during development and on the role of the human PON1192 alloforms in modulating these consequences. The results are relevant to OP sensitivity in humans, because one-half of the U.S. population of Northern European origin is homozygous for the PON1Q192 allele. The experiments examining the effects of maternal PON1 status are providing important information on the relationship of a mother’s PON1 status to her ability to protect a fetus. There is evidence from studies in various populations that children are exposed, either in utero or postnatally, to different OPs. For example, a study in a New York population identified several diethylphosphates (possible metabolites of diazinon or chlorpyrifos, as well as of other OPs) and malathion dicarboxylic acid (a specific metabolite of malathion) in maternal urine [Engel, et al., 2007 Am J Epidemiol 165(12):1397]. The study reported behavioral alterations in newborns, which were associated with the presence of dialkyl-phosphates and appeared to be modulated by PON1 status. Our prenatal exposure experiments with dams of different PON1 status has demonstrated that the PON1-/- dams are less well able to protect their fetuses from CPO exposure.

The biomarker project will provide a much better means of quantifying exposures. For example, it is not possible to determine if an individual has been exposed to chlorpyrifos or its breakdown product trichloropyridinol. The protein biomarkers are generated only by exposure to active OP compounds. The development of mass spectrometry (MS) protocols for determining the percentage modification of biomarker proteins will provide a much more accurate determination of target protein inhibition. Currently, a baseline level of activity is required to establish a reasonable estimate of the percentage inhibition of an individual’s BChE. The MS protocols directly determine the percentage modification without the need for a baseline activity determination. The MS analysis of modified protein should be superior to analysis of urinary metabolites for estimating exposures, since it is not possible to know whether a metabolite was taken up directly from the environment or was generated in vivo. Modification of biomarker proteins happens only when active pesticide or its oxon is taken up by an individual. The methods that we have developed for MS analysis of OP adducts will be important for the MS Facility at CDC (Rudy Johnson, Director) in identifying OP related agents of chemical terrorism and cases of pesticide poisoning. The IMB protocols that we have developed require significantly less than 50 μl of plasma and our preliminary experiments with blood spots indicate that the use of blood spot sample collection, shipping and analysis together with the MS analyses will greatly facilitate exposure studies. It was not possible previously to study and document low level exposures using the inaccurate Ellman activity assay determinations due to both inter- and intra-individual differences in BChE activity levels. The inclusion of APH as a biomarker protein will provide a much longer window in which to evaluate OP exposures and if coupled with analysis of BChE modification together with a series of post-exposure time points should allow the extrapolation back to the time of exposure in cases of acute exposures. Our E. coli protein expression system is allowing us to generate heavy isotope-labeled (15N) recombinant biomarker proteins for use as internal standards in MS analysis of these proteins for our laboratory as well as for CDC’s programs.

The ability to determine whether or not an individual has been exposed to tricresyl phosphate in an airliner cabin as the result of leaky engine seals is very important. Currently, airlines do not inform passengers of any exposures, despite the aircrews often being taken to the hospital at the end of the flight, or in one informative case where the pilot was taken off the airplane on a stretcher and crew members were taken to the hospital in ambulances (see e.g., http://www.telegraph.co.uk/travel/news/fume-incident-hospitalises-american-airlines-crew-and-raises-questions-over-safety-of-cabin-air/). Thus, there is very little information on exposure to children to these toxic fume events, which are estimated to occur with a frequency of one-half percent to one percent of flights. The biomarker research has demonstrated for the first time a physiological response to TCP exposure. Our recent results in analyzing the bioactivation of the meta and para isomers have shown that the active metabolites are different than the CBDP generated from tri-ortho cresyl phosphate. It will be necessary to decorate purified biomarker proteins with the in vitro bioactivated meta and para isomers that will provide adducts with different masses than previously characterized for the tri-ortho isomers.

The relevance of our research on gene-environment interactions involving PON1 and its polymorphisms extends beyond pesticide toxicity to multiple other PON1 substrates, including lactone-containing drugs and prodrugs, quorum sensing factors used by Pseudomonas aeruginosa for virulence and biofilm formation, and oxidized lipids involved in cardiovascular disease and other diseases. Our development of a modified PON1 status assay may allow for the early identification of males who will go on to develop Parkinson’s disease.

Females have higher levels of mitochondrial PON2 and modulate oxidative stress better than males. This finding helps to explain the lower frequency of some neurological diseases in females and provides an understanding as to why we were able to identify 41% of males with PD.

Project-Generated Resources

The protocol for rapid isolation of BChE was transferred to the CDC. We generated anti-human acylpeptide hydrolase antibodies, which also recognize mouse APH. We constructed a plasmid for expressing recombinant NEST (active site domain of neuropathy target esterase). We developed protocols for generating deuterated active OP metabolites from parent compounds, e.g., deuterated CPO from deuterated CPS and deuterated CBDP from deuterated tri-cresyl phosphate. These deuterated compounds are being shared with CDC together with biomarker proteins modified with the heavy isotope analogs. Plasmids and protocols for producing active recombinant human PON1 from an E. coli expression system with two aims 1) structure/function studies and 2) for use in treating OP exposed children. Plasmid and protocol for production of recombinant rabbit PON1 for use in skin and environment decontamination of pesticides or nerve agents.

 

References:

Engel S, Berkowitz G, Barr D, Teitelbaum S, Siskind J, Meisel S, et al. Prenatal organophosphate metabolite and organochlorine levels and performance on the Brazelton Neonatal Behavioral Assessment Scale in a multiethnic pregnancy cohort.  American Journal of Epidemiology 2007;165(12):1397-1404, doi:10.1093/aje/kwm029.


Journal Articles on this Report : 7 Displayed | Download in RIS Format

Other subproject views: All 55 publications 37 publications in selected types All 36 journal articles
Other center views: All 507 publications 224 publications in selected types All 175 journal articles
Type Citation Sub Project Document Sources
Journal Article Inoue S, Becker AL, Kim J-H, Shu Z, Soelberg SD, Weigel KM, Hiraiwa M, Cairns A, Lee H-B, Furlong CE, Oh K, Lee K-H, Gao D, Chung J-H, Cangelosi GA. Semi-automated, occupationally safe immunofluorescence microtip sensor for rapid detection of mycobacterium cells in sputum. PLoS One 2014;9(1):e86018. R834514 (Final)
R834514C004 (2015)
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  • Journal Article Kim DS, Burt AA, Rosenthal EA, Ranchalis JE, Eintracht JF, Hatsukami TS, Furlong CE, Marcovina S, Albers JJ, Jarvik GP. HDL-3 is a superior predictor of carotid artery disease in a case-control cohort of 1725 participants. Journal of the American Heart Association 2014;3(3):e000902. R834514 (Final)
    R834514C004 (2015)
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  • Journal Article Kim DS, Crosslin DR, Auer PL, Suzuki SM, Marsillach J, Burt AA, Gordon AS, Meschia JF, Nalls MA, Worrall BB, Longstreth Jr. WT, Gottesman RF, Furlong CE, Peters U, Rich SS, Nickerson DA, Jarvik GP. Rare coding variation in paraoxonase-1 is associated with ischemic stroke in the NHLBI Exome Sequencing Project. Journal of Lipid Research 2014;55(6):1173-1178. R834514 (Final)
    R834514C004 (2015)
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  • Journal Article Kim DS, Burt AA, Ranchalis JE, Jarvik LE, Eintracht JF, Furlong CE, Jarvik GP. Effects of dietary components on high-density lipoprotein measures in a cohort of 1,566 participants. Nutrition & Metabolism 2014;11(1):44 (9 pp.). R834514C004 (2015)
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  • Journal Article Kim DS, Burt AA, Ranchalis JE, Vuletic S, Vaisar T, Li WF, Rosenthal EA, Dong W, Eintracht JF, Motulsky AG, Brunzell JD, Albers JJ, Furlong CE, Jarvik GP. PLTP activity inversely correlates with CAAD: effects of PON1 enzyme activity and genetic variants on PLTP activity. Journal of Lipid Research 2015;56(7):1351-1362. R834514 (Final)
    R834514C004 (2015)
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  • Journal Article Marsillach J, Suzuki SM, Richter RJ, McDonald MG, Rademacher PM, MacCoss MJ, Hsieh EJ, Rettie AE, Furlong CE. Human valacyclovir hydrolase/biphenyl hydrolase-like protein is a highly efficient homocysteine thiolactonase. PLoS One 2014;9(10):e110054. R834514 (Final)
    R834514C004 (2015)
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  • Journal Article Marsillach J, Becker JO, Vaisar T, Hahn BH, Brunzell JD, Furlong CE, deBoer IH, McMahon MA, Hoofnagle AN, DCCT/EDIC Research Group. Paraoxonase-3 is depleted from the high-density lipoproteins of autoimmune disease patients with subclinical atherosclerosis. Journal of Proteome Research 2015;14(5):2046-2054. R834514 (Final)
    R834514C004 (2015)
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  • Supplemental Keywords:

    RFA, Health, Scientific Discipline, INTERNATIONAL COOPERATION, ENVIRONMENTAL MANAGEMENT, Biochemistry, Environmental Monitoring, Children's Health, Environmental Policy, Biology, Risk Assessment, pesticide exposure, age-related differences, pesticides, children's vulnerablity, biological markers, agricultural community

    Progress and Final Reports:

    Original Abstract
  • 2010
  • 2011 Progress Report
  • 2012
  • 2013 Progress Report
  • 2014
  • Final Report

  • Main Center Abstract and Reports:

    R834514    University of Washington Center for Child Environmental Health Risks Research (2010)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R834514C001 Community-Based Participatory Research
    R834514C002 Pesticide Exposure Pathways
    R834514C003 Molecular Mechanisms
    R834514C004 Genetic Susceptibility