Grantee Research Project Results
2008 Progress Report: An Integrated Computational Framework for the Interpretation of Organophosphorus Pesticide Biomarkers
EPA Grant Number: R833451Title: An Integrated Computational Framework for the Interpretation of Organophosphorus Pesticide Biomarkers
Investigators: Reisfeld, Brad , Lyons, Michael A. , Chambers, Janice E. , Mayeno, Arthur N. , Yang, Raymond S.H.
Institution: Colorado State University , Mississippi State University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 2007 through September 30, 2010
Project Period Covered by this Report: October 1, 2007 through September 30,2008
Project Amount: $748,582
RFA: Interpretation of Biomarkers Using Physiologically Based Pharmacokinetic Modeling (2006) RFA Text | Recipients Lists
Research Category: Hazardous Substance Research Centers , Pesticides , Human Health
Objective:
Although various biomarkers have been used to assess exposure to and poisoning from organophosphorus (OP) pesticides/insecticides, the complexity of OP absorption, distribution, metabolism, and elimination, especially for mixtures of these chemicals, warrants integration of computational modeling tools with the biomarker data for more accurate quantitation and assessment of actual whole body exposures and target tissue dosimetry. The objective of this project is to create a computer-assisted framework to aid in the identification, characterization, and understanding of biomarkers resulting from human exposure to mixtures of OP insecticides, using chlorpyrifos and diazinon as the initial test compounds. The framework will use existing human biomarker data, along with information about population and exposure variability and uncertainty, to reconstruct absorbed dose and exposure scenarios, as well as to predict levels of biomarkers resulting from known exposures to one or multiple OP insecticides.
The aims, expected output, and anticipated outcome of the project have not changed from those described in the original application.
As stated in the original application, the computational tool, the main output, will use human biomarker data to estimate OP levels in target tissues, perform reverse dosimetry to estimate and characterize absorbed doses and exposures for OP insecticides, and evaluate the utility of the various biomarkers. An important additional output of this project will be the in vivo data resulting from targeted intermittent-dose biomarker studies involving each of the two OP insecticides (chlorpyrifos [CPF] and diazinon [DZN]) and their mixture. We expect that the outcome of this effort will be a more accurate approach to the assessment of exposure from OP insecticide mixtures in the cumulative risk assessment process, and therefore, the overall benefit will be an improvement of EPA’s ability to protect public health.
Experimentation Program: The goals of the experimental portions of this project that are being conducted at the Center for Environmental Health Sciences in the College of Veterinary Medicine at Mississippi State University (MSU) have not changed. The experiments are designed to expose rats four times to each of two OP insecticides (CPF and DZN) and to binary mixtures of the two insecticides, and to subsequently determine cholinesterase inhibition in brain and blood and the concentration of the insecticides and their hydrolysis products in blood and urine. Rats will be dosed every fourth day and the experiment will be terminated at 15 days. These data will be provided to the biological modelers at Colorado State University to develop models to estimate exposures based upon reverse dosimetry modeling techniques using the biomarker data. The goal of the initial range-finding experiments was to select dosages that result in measurable cholinesterase inhibition with levels of the insecticides and metabolites that are above the levels of quantification without eliciting toxicological impact.
Modeling Program: The goals of the modeling portions of this project that are being conducted at Colorado State University (CSU) have not changed. The objective remains to create a computer-assisted framework to aid in the identification, characterization, and understanding of biomarkers resulting from human exposure to mixtures of OP insecticides, using CPF and DZN as the initial test compounds. Work to date has focused on several different areas: (i) data mining, (ii) constructing the OP insecticide PBPK models, (iii) improving the simulation core of the software framework, and (iv) developing a graphical user interface for the framework.
Progress Summary:
Work Status
Quality Assurance
As stated in the original application, the major emphases during Year 1 would be on (i) acquiring and vetting pharmacokinetic and variability data from the literature, (ii) developing and testing the physiologically based pharmacokinetic (PBPK) module for the individual OP insecticides and the binary mixture, and (iii) carrying out the dose/exposure optimization experiments and beginning the repeated-dose biomarker studies.
Experimentation Program: Sprague Dawley-derived rats were used in all experiments to select the dosage levels of CPF (7.5 mg/kg body weight) and DZN (60 mg/kg body weight) required to produce the desired brain acetylcholinesterase (AChE) (target tissue) inhibition levels. In preliminary experiments, these dosage levels have given consistent brain AChE inhibition at the target range of 10-20%. These dosage levels resulted in metabolites in the blood and urine that were well above the detection limits. The dosages are higher than the ones suggested in the initial proposal, but achieve the targeted level of inhibition. There was initial concern that the project’s sub-chronic dosage paradigm might result in these lipophilic compounds being sequestered and slowly released and metabolized resulting in an accumulation of compound in the rats and ultimately a substantial rise in AChE inhibition and an unwanted toxic physiological response. This does not appear to be the case, however, with only marginal increases observed in AChE inhibition after the fourth treatment during the 15 day experiment. Two binary mixture dosages also are being used for the two insecticides, the first equal to each of the individual doses delivered concurrently and a lower dosage of one-half the individual compounds delivered concurrently. The higher dosage mixture resulted in AChE inhibition within the anticipated inhibition range, i.e., 40-50%. The first cohort of rats for the data required for the model has been conducted, and the brain AChE inhibition has been determined. The initial data are consistent with the data generated in the preliminary experiments.
Problems were encountered with initial method development for the analytical chemistry portion of the study. Specifically, the identification and quantification of the DZN metabolite 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP) took much longer than anticipated. The IMHP had to be derivatized in order to be analyzed via gas chromatography, a step in the method that greatly increases the time needed for extraction of the analytes. Furthermore, the type of column and temperature program used in the analysis had to be changed in order to detect the IMHP by nitrogen-phosphorus detection. These changes required more effort and consumable supplies than were initially calculated.
Because of the late start of the project and because of the longer time required to optimize dose levels and methodologies, the project is about 3 months behind the intended original schedule.
Modeling Program: Data from the NHANES database for biomarkers associated with OP insecticide exposure have been collected and organized. Work currently is under way to determine the availability of data from other pesticide exposure studies, such as CHAMACOS (Center for the Health Assessment of Mothers and Children of Salinas).
PBPK models for chlorpyrifos disposition have been created in both MCSim and acslXtreme. These models were initially derived from the structure and code created by Timchalk, et al. (Tox. Sci., 66:1, 2002). However, after an examination of various data from the literature, the models were enhanced to include a gastrointestinal (GI) tissue compartment and explicit blood compartments to separate CPF into plasma, protein binding, and cell mass.
The simulation core of the framework uses the software package MCSim. To date, MCSim has been modified in several ways: (i) it can now run under the Microsoft Windows 2000 and XP operating systems, (ii) it now uses a state-of-the-art pseudo-random number generator (Mersenne Twister), (iii) it contains fixes for several computational bugs, and (iv) the build system has been greatly improved for the Linux operating system.
A graphical user interface (GUI) for the software framework is under active development. Thus far, through this GUI, the user can build models, run simulations, and view results in the forward dosimetry mode.
Experimentation Program: The biochemical assays always contain blanks, and results must always be within the range of values that have occurred for years in the MSU laboratory in order to be used. The analytical chemistry assays always contain blanks, spikes and internal standards. Only values above the levels of detection and within the linear portion of the standard curves will be used. Instruments and measuring devices such as pipettors and balances are maintained in calibration.
Modeling Program: Monthly code reviews have been held to assure that the computer codes are correct and that the model structures have been implemented correctly. All of the computer code and revisions are managed and maintained through a Subversion repository using Trac as a front end (https://www.engr.colostate.edu/epastar-trac).
Experimentation Program: Most of the results thus far have involved dose range-finding studies and in method optimization. The first cohort of treated animals has been run and some initial results are presented below.
Table 1 contains the initial data on brain AChE inhibition from the individual compounds and the combination low- and high-dose animals. CPF alone was administered at 7.5 mg/kg body weight and DZN alone at 60 mg/kg. The low dose combination contained 3.25 mg CPF/kg body weight plus 30 mg DZN/kg body weight, and the high dose combination contained 7.5 mg CPF/kg body weight plus 60 mg DZN/kg body weight. Animals were treated on days 0, 4, 8, and 12. Tissue samples on sampling days were taken prior to treatment. The values represent 1-3 replications.
Table 1. Inhibition of rat brain acetylcholinesterase following treatment with chlorpyrifos (CPF), diazinon (DZN), or a high or low binary mixture
Residues below for CPF, DZN, 3,5,6-trichloro-2-pyridinol (TCP), and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP) are reported in parts per million (ppm) in a 1 ml sample of urine. The following chromatograms (Figure 1 to Figure 4) are representative samples for the low- and high-dosage groups at 24 hours post-dose administration and 4 days post-dose administration. The analyte peaks are labeled as IMHP, DZN, CPF, and TCP. The parathion peak is the internal standard used to determine percent recovery.
Figure 1. Low dosage binary mixture 24 hours post dose administration
Figure 2. Low dosage binary mixture 4 days post dose administration
Figure 3, High dosage binary mixture 4 days post dose administration
Figure 4. High dosage binary mixture 4 days post dose administration
Modeling Program: As described earlier, PBPK models have been developed in both MCSim and acslXtreme to describe the disposition of CPF and its metabolites. The current model structure (enhanced compared to that used by Timchalk et al.) is shown in Figure 5.
Figure 5. PBPK model structure
Results of simulations using this model structure with a repeated dosing scenario are shown in Figure 6. In these panels are plotted (a) the AChE in the brain, (b) RBC AChE activity, and (c) CPF levels in the blood over time.
Figure 6. PBPK simulation results
Screenshots of the current version of the graphical user interface for the integrated framework are shown in Figure 7. In these panels are shown some of the elements/screens that have been developed, including (a) one of the screens for parameter entry, (b) a screen to display concentration-time traces, and (c) a screen to display the confidence intervals resulting from Monte Carlo simulations.
Figure 7. Graphical user interface for the modeling framework
Future Activities:
Experimentation Program: Now that conditions have been optimized regarding dosing levels and analytical chemistry techniques (extractions, urine acid hydrolysis conditions, and column run conditions), the experiments are ongoing, and additional replications will be obtained during the second year of the project. In addition, a dose-dependent analysis of the impacts of plasmaprotein binding and RBC binding for the OP insecticides and their metabolites will be undertaken.
Modeling Program: Over the next 6 to 12 months, the base PBPK model for CPF will be extended to DZN and a mixture of CPF and DZN. The framework then will be used in conjunction with the biomarker data from the experiments at MSU to perform Bayesian-based calibration of the PBPK models. In addition, the GUI will be expanded to include the reverse dosimetry mode.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
| Other project views: | All 3 publications | 2 publications in selected types | All 2 journal articles |
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Lyons MA, Yang RS, Mayeno AN, Reisfeld B. Computational toxicology of chloroform: reverse dosimetry using Bayesian inference, Markov chain Monte Carlo simulation, and human biomonitoring data. Environmental Health Perspectives 2008;116(8):1040-1046. |
R833451 (2008) |
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Reisfeld B, Ivy JH, Lyons MA, Wright JM, Rogers JL, Mayeno AN. DoseSim: a tool for pharmacokinetic/pharmacodynamic analysis and dose reconstruction. Bioinformatics 2013;29(3):400-401. |
R833451 (2008) |
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Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.