Grantee Research Project Results
2005 Progress Report: Reducing Uncertainty in Children’s Risk Assessment: Development of a Quantitative Approach for Assessing Internal Dosimetry Through Physiologically-Based Pharmacokinetic Modeling
EPA Grant Number: R830800Title: Reducing Uncertainty in Children’s Risk Assessment: Development of a Quantitative Approach for Assessing Internal Dosimetry Through Physiologically-Based Pharmacokinetic Modeling
Investigators: Bruckner, J. V. , Delp, Michael D. , Bartlett, Michael G. , Fisher, Jeffrey W.
Current Investigators: Bruckner, J. V. , Bartlett, Michael G.
Institution: University of Georgia , Texas A & M University
Current Institution: University of Georgia
EPA Project Officer: Hahn, Intaek
Project Period: February 1, 2003 through January 31, 2007 (Extended to January 31, 2008)
Project Period Covered by this Report: February 1, 2005 through January 31, 2006
Project Amount: $749,991
RFA: Children's Vulnerability to Toxic Substances in the Environment (2002) RFA Text | Recipients Lists
Research Category: Human Health , Children's Health
Objective:
The objective of this research project is to develop and validate a systematic quantitative approach (i.e., a physiologically based toxicokinetic model) for predicting internal dosimetry of deltamethrin (a representative pyrethroid insecticide) in maturing rats, as an animal model for infants and children.
Progress Summary:
Over the past 3 years, many of the aims of this project have been accomplished. It initially was necessary to develop a reliable analytical technique for deltamethrin (DLM) and some of its metabolites to be used in large-scale toxicokinetic (TK) studies. DLM is a relatively potent neurotoxicant in very young animals, so maximally tolerated doses are quite limited. Our TK experiments required blood and tissue samples for an extended period post dosing. DLM levels at later time points are very low; therefore sensitive methods had to be developed to allow accurate quantitation in the small biological sample volumes that could be obtained from neonatal and preweanling rats. Typical time-course TK experiments yield hundreds of samples of blood and eight tissues; that is why it was important to increase substantially the speed of the analyses. Our fully validated high-performance liquid chromatography (HPLC) techniques for DLM should be adaptable to TK and biomonitoring studies of other pyrethroids and pyrethroid mixtures.
Another problem we solved before conducting TK or in vitro metabolism experiments was identification of a suitable vehicle, or diluent. Vegetable (e.g., corn) oil presents problems with delayed/variable gastrointestinal (GI) absorption and poor solubility in aqueous media in vitro. Use of emulsifiers (e.g., Emulphor®) resulted in the trapping of tiny droplets (micelles) of DLM in pulmonary capillaries. Glycerol formal (GF), a vehicle commonly utilized for lipophilic pharmaceuticals, proved superior. Our TK data explain findings by Dr. Kevin Crofton, et al. of EPA’s National Health and Environmental Effects Research Laboratory (NHEERL), who reported DLM to be much more neurotoxic to rats when given in GF rather than Emulphor.® Our experience with GF provided Dr. Mike DeVito, et al., at NHEERL with a vehicle that could be used successfully for pyrethroid experiments in the laboratory.
As relatively little information is available on the GI absorption, systemic disposition and elimination of DLM or other pyrethroids, comprehensive time-course experiments were conducted to delineate the TK of a series of oral doses of DLM in adult rats. DLM appeared to be rapidly but incompletely absorbed from the GI tract. Surprisingly, concentrations in the brain of the highly lipophilic chemical were only one-quarter to one-third of those found in blood. Blood levels diminished more rapidly than brain levels. Another surprising finding was the relatively high levels of DLM in skeletal muscle. Fat accumulated much higher concentrations of DLM, which served to prolong the presence of the insecticide in the body despite its relatively rapid metabolism.
A detailed investigation was conducted to: identify the specific esterase(s) and cytochrome P450s (CYPs) that metabolize DLM in rats; assess the relative contribution of enzymes in the blood and liver to DLM biotransformation; and determine the metabolic rate constants Km and Vmax for each major enzyme. In vitro experiments demonstrated that carboxylesterases (CaEs) were primarily responsible for DLM hydrolysis in plasma and liver. CYP1A2, CYP1A1, and CYP2C11, in decreasing order of importance, oxidized DLM. CYPs-catalyzed metabolism in the liver, as reflected by intrinsic clearance, was more efficient than CaE-mediated metabolism in plasma and liver of adult rats.
The aforementioned blood/organ time-course and metabolic data were utilized to construct a physiologically based pharmacokinetic (PBPK) model for DLM in the adult rat. In vivo tissue:blood distribution ratios, rather than partition coefficients, were employed. About 95 percent of DLM in blood was present in plasma; blood was represented by two subcompartments, plasma and erythrocytes. It was necessary to modify a preliminary model by inclusion of diffusion- rather than flow-limited equations to describe the TK of DLM in the brain, fat, and the slowly-perfused compartment. Provision was made for biliary excretion of unmetabolized DLM. The refined PBPK model’s predictions compared quite favorably with the empirical blood, plasma, fat, and brain profiles for a range of doses and two routes of administration. This will be the first PBPK model to be published for a pyrethroid. It should be adaptable to other pyrethroids and allow internal dosimetry to be forecast for different exposure scenarios.
The next aim was to obtain physiological values for different age groups of immature rats. Newborn pups were sexed and sacrificed when they reached 1, 10, 21, and 28 days of age. Weights of the following organs were measured in each group: liver, spleen, GI tract, kidney, heart, lungs, brain, and fat. These values were combined with values we previously collected for groups of Sprague-Dawley rats up to 280 days old, to produce the most comprehensive organ weight data set currently available for any laboratory animal. A generalized Michaelis-Menten (GMM) body weight growth model was modified and used to predict accurately organ volumes/weights of developing rats of all ages. Different organs exhibited different growth patterns during maturation. Thus, the GMM model is a considerable improvement over the previously-accepted practice of expressing all organ weights as a constant percentage of body weight.
A series of experiments was conducted to characterize the ontogeny of DLM metabolism in maturing male rats. As with the adults, biotransformation was quantified by measuring the rate of disappearance of parent compound from plasma and liver microsomes of postnatal day (PND) 10, 21, 40, and 90 male rats. Intrinsic clearance by liver CYPs and CaEs and plasma CaEs increased substantially with age, because of progressive increases in Vmax.
A parallel TK and neurotoxicity study was carried out in conjunction with the metabolism work. PND 10, 21, 40, and 90 male rats received 10 mg DLM/kg orally. The magnitude of salivation and tremors was scored subjectively for 6 hours. Other groups of rats were sacrificed periodically and levels of DLM and its metabolite 3-phenoxybenzoic acid (3-PBA) were measured. Blood and brain DLM ammonium uranyl carbonates (AUCs) varied inversely with age. Little 3-PBA was produced by PND 10 rats. There was good correlation between neurotoxicity scores and DLM AUCs. These findings provide solid support for Dr. Larry Sheets’ hypothesis that neonatal and preweanling rats’ limited capacity to metabolically inactivate DLM contributes to elevated brain levels and enhanced acute toxicity. The elevated brain levels also may prove to be a factor in developmental neurotoxicity, should preliminary reports of certain pyrethroids’ potential to exert such effects be borne out.
Future Activities:
The only (and most important) remaining goal is to develop and validate a PBPK model for DLM in immature rats. DLM blood and eight tissue time-course data sets have been obtained for four ages of developing rats given three oral doses of DLM. All age-dependent physico-chemical metabolic and physiological values have been derived, with the exception of cardiac output and tissue blood flows. These will be measured with a radiolabeled microsphere technique by Dr. Michael Delp. When these values are available, we will input them and other age-specific parameters into our PBPK model for the adult rat. The simulations of blood and tissue DLM concentrations will be compared with the empirical profiles. In addition, experiments will be carried out to investigate: the role of the immature GI tract and blood-brain barrier in systemic absorption and brain uptake, respectively; whether and to what extent DLM is excreted in the bile; and whether and to what extent DLM is bound to plasma proteins.
Journal Articles on this Report : 7 Displayed | Download in RIS Format
Other project views: | All 29 publications | 12 publications in selected types | All 12 journal articles |
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Anand SS, Bruckner JV, Haines WT, Muralidhara S, Fisher JW, Padilla S. Characterization of deltamethrin metabolism by rat plasma and liver microsomes. Toxicology and Applied Pharmacology 2006;212(2):156-166. |
R830800 (2005) R830800 (2006) R830800 (Final) |
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Anand SS, Kim K-B, Padilla S, Muralidhara S, Kim HJ, Fisher JW, Bruckner JV. Ontogeny of hepatic and plasma metabolism of deltamethrin in vitro: role in age-dependent acute neurotoxicity. Drug Metabolism and Disposition 2006;34(3):389-397. |
R830800 (2005) R830800 (2006) R830800 (Final) |
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Ding Y, White CA, Muralidhara S, Bruckner JV, Bartlett MG. Determination of deltamethrin and its metabolite 3-phenoxybenzoic acid in male rat plasma by high-performance liquid chromatography. Journal of Chromatography B 2004;810(2):221-227. |
R830800 (2003) R830800 (2004) R830800 (2005) R830800 (2006) R830800 (Final) R830900 (2004) |
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Ding Y, White CA, Bruckner JV, Bartlett MG. Determination of deltamethrin and its metabolites, 3-phenoxybenzoic acid and 3-phenoxybenzyl alcohol, in maternal plasma, amniotic fluid, and placental and fetal tissues by HPLC. Journal of Liquid Chromatography & Related Technologies 2004;27(12):1875-1892. |
R830800 (2003) R830800 (2004) R830800 (2005) R830800 (2006) R830800 (Final) R830900 (2004) |
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Kim KB, Bartlett MG, Anand SS, Bruckner JV, Kim HJ. Rapid determination of the synthetic pyrethroid insecticide, deltamethrin, in rat plasma and tissues by HPLC. Journal of Chromatography B 2006;834(1-2):141-148. |
R830800 (2005) R830800 (2006) R830800 (Final) |
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Mirfazaelian A, Kim K-B, Anand SS, Kim HJ, Tornero-Velez R, Bruckner JV, Fisher JW. Development of a physiologically based pharmacokinetic model for deltamethrin in the adult male Sprague-Dawley rat. Toxicological Sciences 2006;93(2):432-442. |
R830800 (2005) R830800 (2006) R830800 (Final) R840032 (2023) |
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Mirfazaelian A, Fisher JW. Organ growth functions in maturing male Sprague-Dawley rats based on a collective database. Journal of Toxicology and Environmental Health Part A 2007;70(12):1052-1063. |
R830800 (2005) R830800 (Final) |
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Supplemental Keywords:
modeling, internal exposure, pharmacokinetics, toxicokinetics, metabolism, toxicology, health effects, human health, risk assessment, susceptible populations, children, pesticides, insecticides, pyrethroids, toxics,, RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, Toxics, ENVIRONMENTAL MANAGEMENT, PESTICIDES, Toxicology, Genetics, Health Risk Assessment, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Environmental Microbiology, Biochemistry, Environmental Monitoring, Physical Processes, Children's Health, genetic susceptability, Pesticide Types, Risk Assessment, health effects, pesticide exposure, pharmacodynamic model, sensitive populations, detoxification, biomarkers, age-related differences, gene-environment interaction, exposure, animal model, developmental effects, metabolic study, children, pharmacokinetic models, insecticides, toxicity, genetic polymorphisms, PBPK modeling, pharmacokinetc model, metabolism, biological markers, risk based model, exposure assessment, organophosphate pesticides, biochemical research, environmental hazard exposures, human health riskRelevant Websites:
http://www.rx.uga.edu/main/home/httpd/html/index.html Exit
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.