Enhancements to Cholinesterase Monitoring: Oxime Reactivation and OP-ChE Adducts

Principal Investigator: Chris Simpson, PhD, MSc
Professor, Environmental and Occupational Health Sciences
University of Washington

NIOSH 2006-2011

Scientists at PNASH and the Centers for Disease Control (CDC) conducted this project to improve the current ChE laboratory test – increasing accuracy to ensure a ChE depression is due to pesticide exposure and identify the specific pesticide involved.

Organophosphorus pesticides (OPs) have widespread commercial application in the United States and worldwide, and their primary toxicological effect is associated with inhibition of cholinesterase (ChE). In Washington State, cholinesterase activity is measured in farmworkers expected to have high exposures to OPs, and when depression in cholinesterase activity is observed, remedial actions are undertaken to reduce exposures and protect worker health. However, the cholinesterase assay lacks sensitivity and specificity, with the result that a substantial number of false-positive and false-negative measurements are obtained. Misclassification of overexposure comes with substantial economic costs to local authorities and growers, and causes unnecessary psychological distress for the farmworkers. In the case of false-negative data, exposure misclassification fails to identify and correct situations where overexposure to pesticides has occurred.

To augment and improve cholinesterase monitoring, we developed and validated two analytical methods designed to measure the interaction of OP pesticides with cholinesterase enzyme. These assays were validated via a series of in vitro studies using human plasma, and in vivo studies in rats and in humans with occupational exposure to OP pesticides. Finally, the assays developed during this project were incorporated into the practice of OP pesticide exposure monitoring in Washington State.

Though not useful for clinical diagnosis, the oxime reactivation assay can be used to help diagnose acute poisoning with OP pesticides and guide treatment – for example, when a subject presents at an emergency clinic with symptoms consistent with pesticide poisoning. The second component of this project is the development of a process for tandem mass spectrometry (HPLC/MS/MS) of protein adducts in the blood of exposed workers. The HPLC/MS/MS method can detect exposures to OP pesticides at low levels more reliably than using the existing cholinesterase activity assays. These more sensitive adduct measurements can be combined with information about personal characteristics and workplace practices, either from surveys or intervention studies, to analyze risk factors associated with pesticide exposure. Results could be used to further educate workers and regulators regarding ways to minimize exposures.

Specific Aims

Aim 1a.  Develop and validate a sensitive, accurate, and robust analytical procedure based on HPLC/MS/MS for the measurement of OP-adducts to plasma ChE (butyryl ChE, BuChE). 

Aim 1b. Evaluate the potential of oxime reactivation followed by measurement of ChE activity for confirmation of depressed ChE activity.

Aim 2. Evaluate the relationships between OP-adduct levels, reactivatability of ChE, and ChE activity in vitro, in vivo in rats, and in humans exposed to OP pesticides.

Aim 3. Research to practice: Incorporate the assays developed in Aim 1 with the practice of OP pesticide exposure monitoring in Washington State.

Partners and Advisories
Organic Analytical Toxicology Branch, National Center for Environmental Health, CDC

Presentation: Monitoring Exposure to Organophosphate Pesticides, 2011 Symposium on Agriculture, Forestry, and Fishing Health and Safety

Measurement of Farmworker OP Exposure through Protein Adducts