Grantee Research Project Results
2000 Progress Report: Physiologically Based Pharmacokinetic Modeling of Haloacid Mixtures in Rodents and Humans
EPA Grant Number: R825954Title: Physiologically Based Pharmacokinetic Modeling of Haloacid Mixtures in Rodents and Humans
Investigators: Schultz, Irvin R. , Corley, Richard A. , Stenner, Robert D. , Bull, Richard J.
Institution: Pacific Northwest National Laboratory
EPA Project Officer: Hahn, Intaek
Project Period: January 1, 1998 through December 31, 2000
Project Period Covered by this Report: January 1, 1999 through December 31, 2000
Project Amount: $536,857
RFA: Drinking Water (1997) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The goals of this project are to: (1) characterize the comparative toxicokinetics and metabolism of chloro, bromo, and mixed chloro-bromo haloacids (HAs) in rodents; and (2) develop a physiologically based pharmacokinetic (PBPK) model, which can predict the tissue distribution and elimination of HAs during chronic oral exposure in mice, rats, and humans. Work during 2000 focused on completing laboratory experiments measuring the biotransformation, low-dose toxicokinetics, and tissue distribution of haloacids (HAs) in rodents.
Excellent progress was made in three areas of the study: completing toxicokinetic and disposition studies of HAs in B6C3F1 mice; collecting experimental data for low dose extrapolation of HAs (using dichloro-acetic acid [DCA] as a representative HA); and improving our understanding of tri-HA metabolism. The experimental methods used in these studies were developed during the first 2 years of the project and are briefly summarized here. For toxicokinetic studies using male F344 rats and B6C3F1 mice, 8- to 10-week old animals were given oral or intravenous doses of a HA. For the low dose extrapolation studies, doses ranged between 0.05 to 20 mg/kg of DCA. All dosing solutions were prepared in 0.9 percent (wt/vol) NaCl (administered volume was 1 mL/kg) and pH adjusted to 7.0 prior to administration. A typical blood sampling schedule after i.v. or gavage dosing was 0, 5, 10, 20, 30, 45, 60, 90, 120, and 180 minutes and, variously thereafter, up to 24 hours. At each sampling time, approximately 0.05 mL of blood was removed and mixed with 0.2 mL of ice cold 0.1 M sodium acetate buffer (pH 5.2) and frozen at -20ºC until later analysis. Actual blood sample volumes were determined gravimetrically using tared vials and assuming blood density was 1.05. Urine and feces were collected after 24 hr from intravenously dosed animals and stored at -20ºC until later analysis.
Experiments studying the in vitro metabolism of tri-HAs used mouse and rat microsomes. Each microsomal incubation typically contained 1.5 mg of microsomal protein, varying concentrations of the tri-HA, and an NADPH regenerating system consisting of 4-12 mM glucose-6-phospate, G-6-P dehydrogenase (0.9 units), and 1.5 mM NADP. The final volume of the incubate was brought to 0.5 mL with 50 mM Kpi (pH 7.4). The incubations were performed under varying oxygen tensions ranging from 0 percent O2 (pure N2 atmosphere), 2 percent O2, and normoxic (normal atmosphere). The samples then were incubated for various times up to 1 hour (depending on the experiment) at 37ºC. The reaction was terminated by denaturing the protein with an organic solvent wash and the samples extracted and analyzed for both the parent and metabolically formed HA.
Progress Summary:
The results of the toxicokinetic and disposition studies in B6C3F1 mice are summarized in two manuscripts that are either in press at the Journal of Applied Toxicology or submitted to Toxicology and Applied Pharmacology. Interested readers should refer to those articles for a complete description of the results. A brief summary of the major findings and conclusions are: in mice, the tri-HAs (using bromodichloro-acetic acid [BDCA] as an example) are metabolized in the liver by both microsomal and cytosolic subcellular fractions. The cytosolic pathway is glutathione dependent and accounted for 78 percent of the total intrinsic metabolic clearance in mice. The enzyme(s) responsible for the cytosolic pathway is unknown, but similarities with the metabolism of DCA suggest a common enzyme for both the di- and tri-HAs. We proposed a mechanism for this cytosolic pathway, where the tri-HA reacts enzymatically with glutathione to form a di-HA-glutathione adduct that decomposes nonenzymatically to oxalyl-glutathione (Ox-GS). The Ox-GS eventually is hydrolyzed enzymatically to free oxalate and GSH. The microsomal metabolism of BDCA in mice accounted for only 22 percent of the total metabolic clearance and proceeded by reductive debromination of BDCA via a dichloroacetate free radical intermediate. The dichloroacetate radical was trapped with the spin-trapping agent PBN and identified by GC/MS. The apparent Km and Vmax for the cytosolic metabolism of BDCA were 212 µM and 0.96 nmol min-1 mg-prot-1, respectively, and 372 µM and 0.79 nmol min-1 mg-prot-1 for the microsomal metabolism. The hepatic clearance of BDCA was determined from the cytosolic and microsomal intrinsic clearances (Vmax / Km) and compared to the in vivo nonrenal clearance in mice. The in vitro hepatic clearance correlated well with the in vivo clearance, with the calculated in vitro clearance overestimating the measured in vivo clearance by only 31 percent.
Additional studies using liver microsomes from male Fischer 344 rats and specific P450 proteins indicate that microsomal metabolism is stimulated under a reducing environment, being highest under pure nitrogen headspace followed by 2 percent oxygen and atmospheric headspaces. The Vmax for the loss of parent tri-HA was four to five times higher under nitrogen headspace than under atmospheric conditions. Intrinsic metabolic clearance for the brominated tri-HAs was of the order tribromo>chloro-dibromo>>BDCA. At high substrate levels, the rate of consumption of the tri-HA was up to two to three times greater than the corresponding rate of formation of the di-HA metabolite (produced via reductive dehalogenation) indicating additional metabolite(s) are being formed. Liberation of free Br- during TBA metabolism corresponded to the expected amount produced after dibromoacetate formation (1:1 stoichiometry). This result indicates the additional metabolite(s) formed does not release Br-. Eadie-Hofstee plots for the consumption of the parent tri-HA appeared linear suggesting a single P450 enzyme is responsible. Carbon monoxide and diphenyleneiodonium (a specific P450 reductase inhibitor) blocked the metabolism of tri-HAs. However, inhibitors of specific P450 proteins (CYP 2E1, 2D6, and 3A4) failed to significantly block metabolism. These experimental results support the hypothesis that cytochrome P450 reductase is primarily responsible for the microsomal metabolism of tri-HAs forming a free radical intermediate, which subsequently undergoes several nonenzymatic reactions leading to the documented metabolites. A summary of the proposed pathways for tri-HA metabolism is shown in Figure 1.
Figure 1. Proposed tri-haloacid metabolism using BDCA as a representative tri-HA DCA= dichloroacetate; OX-GS = oxalyl-glutathione
Toxicokinetic studies of tri-HAs in mice indicate a similar pattern to that observed previously for rats. Elimination of tri-HAs in mice is primarily due to metabolism with first pass hepatic metabolism occurring that can reduce oral bioavailability to less than 75 percent. The volume of distribution (determined after i.v. dosing) appears to be limited to total body water. Nonlinear behavior was exhibited at doses greater than 20 mg kg-1 with a much higher than expected area under the curve (AUC), a decrease in total body clearance (Clb), and an increase in the terminal half-life at the highest doses studied (100 mg/kg).
The bioavailability of HAs at low exposure rates was studied using DCA as a model haloacid. These experiments used both naïve male F344 rats and rats pretreated with DCA to reduce the glutathione-S-transferase-zeta (GSTz) activity. Rats were dosed (intravenously or by gavage) with different concentrations of DCA ranging from 0.05 to 20mg/kg and the blood plasma concentration-time profile characterized. DCA elimination by naïve rats was so rapid only higher doses (1-20 mg/kg for the intravenous dose and 5-20 mg/kg for the gavage) provided values above the analytical limit of detection for DCA. Oral bioavailability was approximately 30 percent in the naïve rats at the 5 and 20 mg/kg doses and zero at doses below 1 mg/kg. Oral bioavailability increased to 39 and 82 percent at 5 and 20 mg/kg doses, respectively in the GSTz depleted rats. Virtually all of the dose was eliminated through nonrenal routes; the urinary clearance was <1 ml h-1kg-1. Comparison of the intravenous dose with the corresponding AUC indicated a lack of linearity across the dose range studied for both naïve and pretreated rats. Table 1 provides a summary of the oral bioavailability of DCA in rats.
Table 1. Oral bioavailability of dichloroacetic acid after oral administration of a range of doses in naïve and GSTz depleted adult male F344 rats
Future Activities:
Work during the upcoming year will concentrate on PBPK model development and preparation of manuscripts.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 31 publications | 10 publications in selected types | All 10 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
Merdink JL, Bull RJ, Schultz IR. Trapping and identification of the dichloroacetate radical from the reductive dehalogenation of trichloroacetate by mouse and rat liver microsomes. Free Radical Biology and Medicine 2000;29(2):125-130. |
R825954 (2000) |
|
|
Merdink JL, Bull RJ, Schultz IR. Toxicokinetics of bromodichloroacetate in B6C3F1 mice. Journal of Applied Toxicology 2001;21(1):53-57. |
R825954 (2000) |
Exit Exit |
Supplemental Keywords:
drinking water, exposure, risk, enzymes, bioavailability, metabolism, mixtures, halogenated acetic acids, toxicology, toxicokinetics, mass spectroscopy., RFA, Scientific Discipline, Water, Environmental Chemistry, Health Risk Assessment, Analytical Chemistry, Drinking Water, halogenated disinfection by-products, tissue distribution, biomarkers, human health effects, exposure and effects, chemical byproducts, disinfection byproducts (DPBs), dose response, exposure, pharmacokinetics, haloacetic acids, PBPK modeling, treatment, metabolism, drinking water contaminants, drinking water system, haloacids, toxicokinetics, rodents, enzyme inactivationProgress 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.