Molecular Recognition in Biological Systems and Bioinformatics

CYP-Specific PBPK/PD Models to Interpret Biomarkers for Organophosphate Pesticides.

Assessing the Health Risks of Exposures to Organophosphate (OP) Pesticides; Utilize enzyme-specific physiologically based pharmacokinetic /pharmacodynamic (PBPK/PD) models to better assess human exposure, target tissue dose and subsequent effects of organophosphate pesticides (OPs); Identifying new biomarkers of susceptibility to this class of pesticides by investigating the function of genetic variants in key enzymes (CYP2B6, CYP2C19, PON1) which regulate OP metabolic activation and detoxification; Assessing the Biological and Toxicological Effects of Exposures to Persistent Halogenated Aromatic Hydrocarbons, including Dioxins, Polybrominated Diphenyl Ethers (PBDEs) and Polychlorinated Biphenyls (PCBs); Assessing Human Exposures to dioxins, PBDEs, and PCBs; Metabolism and disposition of dioxins, PBDEs, and PCBs; Utilizing toxicogenomic approaches to understand the relationship between exposures to dioxins and/or PCBs and mechanisms for adverse heath effects; Identifying alterations in global gene expression and specific pathways that may be associated with pathological responses of AhR ligands (dioxins, PCB126) and non- AhR ligands (PCB153); Characterizing the adverse effects of dioxin on lung development and the role of the AhR pathway in the developing lung.

CYP-Specific PBPK/PD Models to Interpret Biomarkers for Organophosphate Pesticides

Description:

(a) Objectives: The primary objective of the proposed studies is to improve existing models to better estimate exposures, target tissue dose and resulting effects in human populations, utilizing the abundance of urinary metabolite/biomarker data for the organophosphate (OP) pesticides, chlorpyrifos, parathion, methyl parathion, and diazinon. Current physiologically based pharmacokineticpharmacodynamic (PBPK/PD) models, which utilize single kinetic constants (Km, Vmax) for a given metabolic pathway, using rat liver microsomes, do not accurately reflect human enzymes or the variability in content and activity of key enzymes (cytochrome P450s, CYPs; paraoxonase-1, PON1) which regulate the metabolic activation and detoxification of OPs in human populations exposed to pesticides. It is hypothesized that more accurate measures of exposure, target tissue dose and subsequent effects will come from existing PBPK/PD models, which incorporate human CYP-specific kinetic parameters (Km and Vmax) for OP metabolism, CYP–specific content in the liver, and the function and content of genetic variants in key enzymes (CYP2B6, CYP2C19, PON1) which regulate OP metabolic activation and detoxification.

(b) Approach (Specific Aims): 1. Generate CYP-specific PBPK/PD models, utilizing existing urinary metabolite / biomarker data for chlorpyrifos, parathion, methyl parathion, and diazinon, to better estimate exposure, target tissue dose and effects of these OPs in human populations by incorporating 1) kinetic parameters (Km and Vmax) for OP metabolism by specific human CYPs, 2) hepatic content of specific CYPs, 3) PON1 activity and content, and 4) serum protein binding data for OPs and metabolites into existing PBPK/PD models. 2. Generate the kinetic parameters for OP metabolism by prevalent polymorphic isoforms of CYP2B6, CYP2C19 and PON1, which are known to exhibit altered enzyme activity and/or expression (biomarkers of susceptibility). 3.Integrate the altered activity and expression data from the key polymorphic CYPs and PON1 (aim 2) into the CYP-specific PBPK/PD models (aim 1). 4. Incorporate the data from aims 1-3 into a multiroute, multichemical, PBPK/PD model (ERDEM, Exposure Related Dose Estimating Model) that can convert urine biomarker data for several OP pesticides to measures of exposure, target tissue dose and effect (inhibition of acetylcholinesterase) in populations of interest.

(c) Expected Results: Since the hepatic levels of specific CYPs exhibit marked variability across both population and age groups, the proposed CYP-specific PBPK/PD models should prove to be more accurate and more easily modified to address factors such as age, CYP content and genetic polymorphisms in CYPs and PON1 (biomarkers of susceptibility). CYP-Specific PBPK/PK models for OP pesticides, that better estimate population variability in exposure, target tissue dose and effect, will be valuable tools for risk assessment efforts for these OPs in the general population, special exposure groups, susceptible individuals (polymorphisms in CYPs and /or PON1), and 6-11 year old children, which are the age group in the U.S. with the highest levels of urinary metabolites specific for these commonly used OPs (CDC 2005).

Biomarkers of Organophosphate Pesticide-Induced Neurotoxicity

Project Summary/Abstract:

Organophosphorus pesticides (OPs) are the most commonly used pesticides in the U.S. and worldwide. Evidence from human and animal studies clearly identifies neurotoxicity as the primary endpoint of concern. However, it has been difficult to predict the risk that repeated low-dose exposure to OPs pose to humans because: 1) a relationship between OP dose and neurobehavioral deficits has yet to be established in humans; 2) biomarkers that reliably predict OP-induced neurobehavioral deficits are not available: and 3) the potential for genetic variation to modify exposure sensitivity has not been thoroughly investigated. The proposed studies will test the hypotheses that OP-induced neurobehavioral deficits are dose-related and that measures of oxidative stress and inflammation are better predictors of neurobehavioral deficit than cholinesterase inhibition. These hypotheses will be tested by studying a cohort of pesticide application workers in Egypt’s Menoufia Governorate previously reported to exhibit the broadest range of neurobehavioral deficits in humans following OP exposure. This Egyptian cohort is uniquely suited for these studies because, unlike most pesticide exposures, the exposure is simple (a single OP, chlorpyrifos) and consistent within job categories, but with substantial differences between job categories. In aim 1, OP doses will be estimated using PBPK/PD modeling of urinary OP metabolite data collected from 255 Egyptian workers over the application cycle. These workers will also be genotyped for polymorphisms of key enzymes involved in OP metabolism (CYP2B6, CYP2C19 and PON1) to evaluate the potential for genetic variation to modify internal dose. In aim 2, behavioral deficits will be determined in a subset of workers exhibiting a range of OP exposures. Data from aims 1 and 2 will be integrated to determine the relationship between OP dose and neurobehavioral deficits. Rat studies will be conducted in parallel (aim 3) to test candidate biomarkers as predictors of OP-induced neurobehavioral deficits. The specific biomarkers that will be examined include cholinesterase inhibition, urinary isoprostanes as a measure of oxidative stress, and serum levels of C-reactive protein and inflammatory cytokines as measures of inflammation. In aim 4, those biomarkers that predict OP-induced neurobehavioral deficits in rats will be tested to determine if they similarly predict deficits in behavioral performance in Egyptian pesticide workers. The proposed studies will provide critical data needed to develop effective biomarkers of OP exposure, biological response and genetic susceptibility. The availability of such biomarkers would facilitate the identification of at-risk individuals as well as the testing of intervention and treatment strategies, and the need to develop these strategies is underscored by evidence of widespread human exposure to OPs and the credible threat of OPs as chemical agents of terrorism.

Rat, mouse, human

AhR signaling pathway, CYPs, PON1

Human exposure to persistent organic pollutant and OP pesticides, and health risks associated with these exposures.

qRT-PCR, micro array, western blotting, enzyme activity, restriction fragment length polymorphism (RFLP)

AhR reporter gene assay

HepG2, MCF7/MX100, hepa 1c1c7

HPLC, GC/ECD, GC/MS

Cancer, liver injury, lung development

Lung, liver

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