Grantee Research Project Results
2006 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, 2006 through January 31, 2007
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:
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:
Most of the aims of the project have been accomplished. It was initially necessary to develop a reliable analytical technique for deltamethrin (DLM) and some of its metabolites that could 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 require blood and tissue samples for an extended period post dosing. DLM levels at later time-points are very low, so sensitive methods had to be developed that would allow accurate quantitation in the small biological sample volumes that are obtained from neonatal and preweanling rats. Typical time-course TK experiments yield hundreds of samples of blood and eight tissues, so it was important to substantially increase 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 to solve before conducting TK or in vitro metabolism experiments was identification of a suitable vehicle or diluent for DLM. 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 trapping of tiny droplets (micelles) of DLM in pulmonary capillaries. Glycerol formal (GF), a vehicle commonly utilized for lipophilic pharmaceuticals, proved superior. The oral bioavailability, brain levels, and severity of neurotoxicity were all greater in the rats given DLM in GF. These data explain findings of Dr. Kevin Crofton et al., of the National Health and Environmental Effects Research Laboratory (NHEERL), who reported DLM to be 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 they have since used successfully with pyrethroid experiments in their laboratories.
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. Oral bioavailability was only 15–18%. Surprisingly, concentrations of the highly lipophilic chemical in the brain were only ¼–⅓ of those in blood. Blood levels, however, diminished more rapidly than brain levels. Another surprising finding was relatively high levels in skeletal muscle. Fat and skin accumulated even higher concentrations, which served to prolong the presence of the insecticide in the body, despite its relatively rapid metabolism. The terminal elimination half-lives in these tissues ranged from 150–200 hours.
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 to 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 PBPK model for DLM in the adult rat. In vivo tissue:blood distribution ratios, rather than partition coefficients, were employed. About 85% of DLM in blood was present in plasma, so blood was represented by two subcompartments, namely 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. 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 is the first PBPK model to be published for a pyrethroid. It should be adaptable to other pyrethroids, so their internal dosimetry can be forecast for different exposure scenarios and utilized in risk assessments.
The next specific 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 in order 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 accurately predict organ volumes/weights of developing rats of all ages. Different organs exhibited different growth patterns during maturation. The GMM model is a considerable improvement over the common practice of expressing 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 adult, 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, due to progressive increases in Vmax. CYPs- and CaEs-mediated biotransformation peaked in PND 21 and 40 rats, respectively.
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) measured. Blood and brain DLM area-under-the-concentration (AUCs) varied inversely with age. Little 3-PBA was produced by PND 10 rats. There was good correlation between neurotoxicity scores and brain DLM AUCs. These findings provide 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 of high doses of the pyrethroid. Relatively minor age-related differences, however, were observed at the lowest DLM dose. The elevated brain levels at high dosages may prove to be a factor in developmental neurotoxicity should preliminary reports of certain pyrethroids’ potential to exert such effects be borne out.
A subsequent in vitro experiment was conducted to assess both age- and gender-dependent differences in DLM metabolism by S-D rat liver and plasma. There was no gender difference in PND 21 animals, but intrinsic clearance was higher in the adult males. Groups of PND 21 and 90 rats of each sex were given 10 mg DLM/kg orally (p.o.). All PND 21 rats died. No gender disparity was evident in adults dosed with 10 mg/kg, but females given 20 mg/kg exhibited more pronounced salivation and tremors than did males. Thus, the lesser capacity of adult female rats to metabolically-detoxify DLM was manifest only at a quite high dose. The ontogeny and regression of isozymes and enzymes that metabolize DLM were characterized in female rats ranging in age from 5 days to 15 months. Activities of hepatic CYP1A1/2, 2B1/2, and 2E1, as well as hepatic and plasma CaEs and aryl esterases, peaked on days 21 or 40. Little regression in these enzymes’ activities was seen in the 15-month-old female rats.
In light of the low oral bioavailability of DLM and its very modest presence in the brain (0.1–0.3% of absorbed doses), the ability of the brain, intestine, and lung to metabolize DLM was examined. CYP-mediated metabolism was not observed in any of the tissues. No CaE-catalyzed metabolism occurred in intestinal samples. Intrinsic clearance by CaEs in lung was 2-fold higher than in brain, though these clearance values were 25- to 50-fold lower than in the liver. Thus, it can be concluded that intestinal and lung metabolism have little role in first-pass elimination of oral DLM, and that the contribution of DLM metabolism in the brain to very low levels there is minimal.
The third and final specific aim of the project was to characterize the dose-dependence of DLM TK in maturing rats and to utilize these data to develop and validate a PBPK model for immature rats. Serial blood and tissue samples were taken for up to 96 hours from PND 10, 21, 40, and 90 rats given 0.4, 2, or 10 mg DLM/kg p.o. and analyzed for their DLM content. The resulting time-course data were used to calculate classical TK parameters, as well as indices (e.g., in vivo tissue:blood distribution ratios) for a PBPK model. Work was begun to construct a PBPK model for DLM for immature S-D rats.
Future Activities:
The only remaining goal is development and validation of a PBPK model for DLM in immature rats. DLM blood and eight tissue time-course data sets have been obtained for three ages of developing rats given three oral doses of DLM. All age-dependent physico-chemical, metabolic, and physiological values have been derived experimentally, 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. Then simulations of blood and tissue DLM concentrations will be compared with the empirical profiles. If funding allows, experiments will also be carried out to investigate: the rate and magnitude of DLM absorption from the stomach and the intestine; the roles of the immature GI tract and blood-brain barrier in systemic absorption and brain uptake, respectively; the potential role of P-glycoprotein in efflux of DLM from the brain and GI tract; and whether and to what extent DLM is excreted in the bile and bound to plasma proteins. Knowledge of these processes would make it possible to develop more physiologically correct models that can provide more accurate simulations of systemic and central nervous system (CNS) dosimetry.
Journal Articles on this Report : 8 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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Kim K-B, Anand SS, Muralidhara S, Kim HJ, Bruckner JV. Formulation-dependent toxicokinetics explains differences in the GI absorption, bioavailability and acute neurotoxicity of deltamethrin in rats. Toxicology 2007;234(3):194-202. |
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, Kim K-B, Lee S, Kim HJ, Bruckner JV, Fisher JW. Organ growth functions in maturing male Sprague-Dawley rats. Journal of Toxicology and Environmental Health-Part A 2007;70(5):429-438. |
R830800 (2006) 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.