2011 Progress Report: Genetic SusceptibilityEPA 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
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 1, 2010 through July 31,2011
RFA: Children's Environmental Health and Disease Prevention Research Centers (with NIEHS) (2009) RFA Text | Recipients Lists
Research Category: Children's Health , Health
- Recently, we added two additional potential biomarkers of toxicity and/or exposure, monocyte carboxylesterase (CES) and neuropathy target esterase (NTE), that are present in blood. The OP modification of BChE was initially determined by incubating plasma with 25 μM chlorpyrifos oxon (CPO) and 25 μM azinphos methyl oxon (AZO) to generate the ethyl and methyl modified active site serines. In both cases, the OP adducts aged to the mono acyl derivatives, mono-ethyl phosphoserine in the case of CPO and mono-methyl phosphoserine in the case of APMO. The mono-methyl phosphate added 94 Da to the active site peptide (chymotryptic digest) GES*AGAASVSLH, while the mono-ethyl phosphate added 107 Da to the active site peptide. We currently are determining the limit of detection of modified peptide by injecting different ratios of modified vs. unmodified peptide digests into the mass spectrometer (MS). A ThermoFisher LTQ-FT with a focusing lens was installed in the MS facility increases the sensitivity of the instrument by at least 5-fold.
- We currently are characterizing the CPO and APMO adducts of recombinant and red cell APH. The red cell APH has a much longer half-life than the plasma BChE. We have identified the peptide modified by OP exposure by mass spectrometry (collaboration with Dr. Mike MacCoss). We also have generated 15N-labeled standard APH by expression of APH in an E. coli system. The 15N-labeled APH will be used for quantifying the RBC APH modified by exposure. We have added two additional target biomarker proteins, CES1 and NTE, to the panel of OP biomarker target proteins. These proteins should be accessible from the mononuclear cell fraction of blood, and also, in the case of NTE, from platelets. We characterized CES activity in an activity stained gel by analyzing an extract from a cultured human monocyte cell line. We have generated recombinant CES1, using a baculovirus expression system, and are in the process of purifying this protein. For assessment of NTE as a biomarker, we are cloning the active-site peptide, NTE esterase domain (NEST), into an expression system. Commercial antibodies that recognize human and mouse NTE also are available.
- The generation of anti active-site peptide antibodies is under way both for APH and BChE. We think that having very pure target active site peptide will allow for a more rapid identification of unusual modifications and thereby a better characterization of unusual exposures to ChE inhibitors. We currently are optimizing the isolation protocols for APH from the brains and red cells of OP-exposed mice. The optimization protocols that we developed during this past year for BChE, which included the use of special tubes and high temperature wash of the immunomagnetic beads to remove loosely bound antibodies, will facilitate the generation of highly pure APH. The two-step IMB protocol, where we first isolate highly purified target protein (BChE and APH), then follow with the digestion of the purified target protein and isolate the active-site peptides in a second-step IMB purification, should produce a nearly pure modified and unmodified target active site peptide. This will facilitate the characterization of unusual adducts to the active site serine. We are transferring these protocols to Rudy Johnson at CDC. These same biomarkers are being used to assess exposure to OP-related compounds in aircraft-cabin air. Modification of BChE by exposure to OP-related compounds in engine oil leaks in aircraft cabin air was determined by incubation of BChE with the chemically synthesized active metabolite of tricresyl phosphate (TCP), CBCP or cyclic saligenin cyclic phosphate. While the TCP does not inhibit BChE, its toxic metabolite 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP) is a very potent inhibitor. A manuscript describing the CBCP was published in 2010 (Schopfer, et al., 2010). Several adducts were observed; the 170 Da cresyl phosphoserine, a 186 Da adduct where the cresyl group was lost and the open ring saligenin was bound and an unusual aged adduct phosphate (+ 80 Da) where all acyl groups were lost. This is, to our knowledge, the first example of an aged organophosphate adduct where all of the acyl groups are lost on aging. In addition, a 156 Da adduct was observed, which would be consistent with a phenyl phosphoserine adduct as observed when we inhibited BChE in vitro with phenyl saligenin cyclic phosphate. To set the magnetic bead protocol up for high-throughput, a magnet was fabricated that allows processing of 96 samples in a standard microtiter plate. We have been able to use rat liver microsomes to bioconvert TCP to active esterase inhibitor(s) in vitro, and are using this technique to inhibit BChE for detection of adducts by MS. Surprisingly, tri-paracresyl phosphate also converts to an inhibitor of some esterases in vivo. The anti-human APH antibodies that we generated also recognize mouse APH.
- Working on methods for isolation of BChE from mouse plasma, which will require the use of a different antibody than the one currently used for isolation of human BChE. We are raising antibodies against CES, and purchasing anti-NTE antibodies, which will be useful for isolating these enzymes from mouse tissues.
- For the experiments characterizing PON1 status as a biomarker of sensitivity to chlorpyrifos (CP) and CPO during gestation, the initial chronic dose-response studies in pregnant and non-pregnant 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 these 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. Continuing studies are using chronic gestational exposure in these dose ranges to determine the importance of PON1 status on maternal and fetal health in mice of all four PON1 genotypes. Recent FISH analyses revealed that the transgenic mice carry multiple copies of the transgenes on different chromosomes, which probably explains at least some of the variability in PON1 levels observed in pups from the same breeding pairs of mice. Mice now are being backcrossed to the PON1-/- background to elucidate the effect of copy number on PON1 activity levels, which we anticipate will produce “low expressers” and “high expressers” with lower interindividual variability.
Journal Articles:No journal articles submitted with this report: View all 55 publications for this subproject
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
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