Measurement of Farmworker OP Exposure through Protein Adducts

This project developed an assay to improve understanding of worker exposures to a wide range of organophosphate (OP) pesticides and advanced the method for potential use in field and clinic settings, providing rapid feedback to workers, clinicians, and physicians.
Organophosphorus pesticides (OPs) have widespread commercial application in the U.S. and worldwide. Although organophosphorus pesticides (OPs) continue to be used in agriculture, there is considerable concern regarding the adverse health effects of these compounds on agricultural workers as well as the general population. Our previous work developed an assay that provides greater sensitivity and specificity as compared to traditional cholinesterase activity monitoring – eliminating the need for collection of a baseline pre-exposure blood sample from each worker. This research study expands this assay to improve the understanding of worker exposures to a wide range of OP pesticides and further developed it for potential use in field and clinic settings, providing rapid feedback to workers, clinicians, and physicians.


Specific Aims

Aim 1. Develop a sensitive mass spectrometric (MS)-based assay that can provide an accurate determination of the percentage modification of the active site serine residues of plasma butyrylcholinesterase (BChE) and red blood cell
(RBC) acylpeptide hydrolase (APH), eliminating the need for a determination of baseline enzymatic activity values for workers prior to the spraying season.
Aim 2. Generate antibodies specific to native and OP-modified BChE to be used in assays to detect OP exposure. 
Aim 3. Adapt the IMB-MS assays for OP modified BChE and APH developed in Aim 1 for application to dried
blood spots (DBS).
Aim 4. Utilize the IMB/HPLC-tandem-MS and immunoassays to measure OP exposures in blood samples obtained from occupationally exposed farmworkers. 
Researchers expanded their previously developed OP-adduct assay to include other protein targets in addition to ChE: acylpeptide hydrolase (APH) and carboxylesterase (CE). These additional enzymes are more sensitive than butyrylcholinesterase (BChE) to certain OP pesticides, increasing the breadth of OP pesticide exposures that can be monitored. Progress was made towards an immunomagnetic bead (IMB) based purification and LC-MS/MS assay to include other protein targets in addition to BChE (i.e. acylpeptide hydrolase (APH) and carboxylesterase (CE)). Antibodies were obtained for purification of these protein targets. We demonstrated the effectiveness of the IMB purification in whole blood, plasma, and dried blood spots. However, work remains to optimize the method.
Another improvement in the previous method included decreasing the amount of blood required such that a dried blood spot (DBS) can be analyzed. This could allow monitoring using a simple finger-stick rather than a blood draw to provide a rapid and complete understanding of worker exposures to a wide range of OP pesticides. We have demonstrated that we can recover and identify BChE and APH from DBS. Our focus on defining the sensitivity and reproducibility of measurement of chlorpyrifos adducts to BChE and APH in DBS. Experiments to define the sensitivity and reproducibility of measuring chlorpyrifos adducts to BChE and APH in DBS are not yet completed.
As part of the project, we developed a biosample repository containing ~350 blood samples obtained from pesticide exposed workers in Washington State, and in Pakistan. These populations represent a range of levels in pesticide exposure and can be used by PNASH researchers, and other colleagues, to evaluate the performance of field deployable assays of OP exposure. Work continues to optimize the on-bead digestion and LC-MS/MS analysis steps for the IMB-based assays, in both venipuncture and DBS samples. When this optimization is complete the optimized assays will be applied to samples in our biorepository to evaluate assay performance.
Principal Investigator: Chris Simpson, PhD, MSc
Professor, Environmental and Occupational Health Sciences
University of Washington
NIOSH 2011-2016 and Washington State MAAF 2012-2013
Partners and Advisories
Central Washington Occupational Medicine Clinic
The Healthy Worker Clinic 
National Institute for Biotechnology & Genetic Engineering 
Yang M, Zhao Y, Wang L, Paulsen M, Simpson CD, Liu F, Du D, Lin Y. Simultaneous detection of dual biomarkers from humans exposed to organophosphorus pesticides by combination of immunochromatographic test strip and ellman assay. Biosens Bioelectron. 2018 May 1;104:39-44. doi: 10.1016/j.bios.2017.12.029. Epub 2017 Dec 21. PubMed PMID: 29306031; PubMed Central PMCID: PMC5794565.
Krenz JE, Hofmann JN, Smith TR, Cunningham RN, Fenske RA, Simpson CD, Keifer M. Determinants of butyrylcholinesterase inhibition among agricultural pesticide handlers in Washington State: an update. Ann Occup Hyg. 2015 Jan;59(1):25-40. doi: 10.1093/annhyg/meu072. Epub 2014 Sep 26. PubMed PMID: 25261454; PubMed Central PMCID: PMC4290628.
Searles Nielsen S, Checkoway H, Zhang J, Hofmann JN, Keifer MC, Paulsen M, Farin FM, Cook TJ, Simpson CD. Blood a-synuclein in agricultural pesticide handlers in central Washington State. Environ Res. 2015 Jan;136:75-81. doi: 10.1016/j.envres.2014.10.014. Epub 2014 Nov 20. PubMed PMID: 25460623; PubMed Central PMCID: PMC4548290.
Searles Nielsen S, Hu SC, Checkoway H, Negrete M, Palmández P, Bordianu T, Racette BA, Simpson CD. Parkinsonism Signs and Symptoms in Agricultural Pesticide Handlers in Washington State. J Agromedicine. 2017;22(3):215-221. doi: 10.1080/1059924X.2017.1317684. PubMed PMID: 28418778; PubMed Central PMCID: PMC5533575.