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Final 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. , Bull, Richard J. , Corley, Richard A. , Stenner, Robert D.
Institution: Battelle Memorial Institute, Pacific Northwest Division
EPA Project Officer: Nolt-Helms, Cynthia
Project Period: January 1, 1998 through December 31, 2000
Project Amount: $536,857
RFA: Drinking Water (1997)
Research Category: Drinking Water
Description:
Objective:The objectives of this project were to: (1) characterize the comparative pharmacokinetics of chloro, bromo, and mixed chloro-bromo haloacids (HAs) in rodents and human tissue; and (2) develop a physiologically based pharmacokinetic (PBPK) model for prediction of tissue distribution and elimination of HAs during chronic oral exposure in mice, rats, and humans.
Summary/Accomplishments (Outputs/Outcomes):When this project commenced activity, relatively little was known regarding the disposition of brominated and mixed bromo-chloro HAs. Therefore, the initial focus of the project examined the comparative toxicokinetics of these compounds in F344 rats, and gathered the data necessary for future construction of PBPK models. In these experiments, rats were administered a 500 µM/kg dose of an HA by intravenous (i.v.) injection or oral gavage. Additional experiments measured blood/plasma partitioning and plasma protein binding. The results of this study were published in Schultz, et al. (1999). Major findings conclude that the disposition of HAs can be broadly grouped into three categories: (1) trichloroacetate (TCA), the most biologically persistent HA, exhibiting low metabolism and moderate urinary excretion and plasma protein binding; (2) di-haloacids, which are highly metabolized and undergo low urinary excretion and are not bound to plasma proteins; and (3) brominated tri-haloacids, which are moderately bound to plasma proteins and are intermediate in character between TCA and the di-HAs, exhibiting both high metabolism and urinary excretion. In later studies of tissue, blood partitioning indicated the ratio of HA concentration in most tissues (liver, kidney, muscle, lung) to that in blood varied little, and was between 0.5 - 0.9. These results indicated that simplifying assumptions of both uniform tissue distribution and tissue partitioning (tissue/blood ratios) for the di- and tri-HAs can be made in PBPK modeling without significantly affecting model output. Furthermore, the importance of metabolism in determining the persistence of HAs was identified, and greater emphasis was placed in future studies on understanding the biotransformation of HAs.
The disposition of dichloroacetate (DCA) and TCA was studied in B6C3F1 mice to better understand whether prior exposure to these HAs alter their metabolism. In this study, published by Gonzalez-Leon, et al. (1999), B6C3F1 mice were given DCA or TCA in their drinking water at 2 g/L for 14 days, and then challenged with a 100 mg/kg i.v. (non-labeled) or gavage (14C-labeled) dose of DCA or TCA. The challenge dose was administered after 16 hours fasting and removal of the HA pretreatment. The DCA or TCA blood concentration-time profiles and the disposition of 14C were characterized and compared with controls. The effect of pretreatment on the in vitro metabolism of DCA in hepatic S10 also was evaluated. The results of this study indicated prior treatment with DCA had a significant affect on the disposition and toxicokinetics of subsequent doses of DCA, but little affect on the disposition of subsequent doses of TCA. Pretreatment with TCA has a minimal effect on the metabolism or toxicokinetics of subsequent challenge doses of TCA or DCA in B6C3F1 mice. Thus, a significant finding indicated that prolonged exposure to DCA (a di-HA) but not TCA (a tri-HA) altered metabolism and elimination rates in rodents.
During the time period of this project, the enzyme responsible for DCA metabolism was identified by other researchers to be glutathione-S-transferase zeta (GST-zeta). DCA also is a mechanism-based inhibitor of GST-zeta. A previous study by our group demonstrated that a loss in GST-zeta enzyme activity occurs after prolonged drinking water exposures. GST-zeta also is known as maleylacetoacetate isomerase (MAA), and is a component of the phenylalanine/ tyrosine catabolism pathway. This connection to tyrosine catabolism was deemed significant, as human genetic deficiencies in other enzymes of the catabolism pathway are associated with increased cancer risk. The improved understanding of DCA metabolism and its connection to tyrosine catabolism warranted more thorough examination of DCA disposition in mice, and is reported in Schultz, et al. (2002). In this study, the internal dosimetry of DCA during prolonged drinking water exposures was measured in mice serially sampled during a 24-hour light-dark cycle. During this 24-hour light-dark cycle, individual mice were exposed to graded drinking water concentrations of DCA. Additional experiments measured the toxicokinetics, in vitro metabolism of DCA and GST-zeta immunoreactive protein levels in the livers of mice given various DCA treatments.
We also measured the MAA isomerase activity in liver cytosol from treated mice
to directly test the hypothesis that DCA exposure decreases. We performed these
experiments using young (8-10 weeks old) mice or in some instances, included
aged/senescent mice (60 weeks old) to study the influence of age and exposure
duration on the disposition of DCA. Results indicated young mice were the most
sensitive to changes in DCA elimination after drinking water treatment. The
in vitro metabolism of DCA was decreased at all treatment rates. Partial
restoration (~ 65 percent of controls) of DCA elimination capacity and hepatic
GST-zeta activity occurred after a 48-hour recovery from 14 d 2.0 g/L DCA drinking
water treatments. Recovery from treatments could be blocked by interruption
of protein synthesis with actinomycin D. MAA isomerase activity was reduced
by more than 80 percent in liver cytosol from 10-week-old mice. However, MAA
isomerase was unaffected in 60-week-old mice. These results indicate that in
young mice, inactivation and re-synthesis of GST-zeta is a highly dynamic process,
and that exogenous factors that deplete or reduce GST-zeta levels decrease DCA
elimination and may increase the carcinogenic potency of DCA. As mice age, the
elimination capacity for DCA is less affected by reduced liver metabolism. Mice
appear to develop some toxicokinetic adaptation(s) to allow elimination of DCA
at rates comparable to naive animals. Reduced MAA isomerase activity alone is
unlikely to be the carcinogenic mode of action for DCA, and may in fact only
be important during the early stages of DCA exposure. The nonlinear aspect of
HA toxicokinetics makes it important to establish the dose range over which
the assumption of linear kinetics can be applied. This is particularly important
for low-dose extrapolation procedures that attempt to extrapolate results from
rodent studies using comparably high exposure levels, to the much lower levels
humans receive from municipal drinking water supplies. The project addressed
this problem by studying DCA kinetics in rats at multiple dose levels (Saghir
and Schultz, 2002). In this portion of the project, F344 rats were administered
(i.v or gavage) graded doses of DCA ranging from 0.05-20 mg/kg and time-course
blood samples collected from the cannula. GST-zeta activity was depleted by
exposing rats to 0.2 g/L DCA in drinking water for 7 days prior to initiation
of toxicokinetic studies. Results indicated that elimination of DCA by naive
rats was so rapid that only 1-20 mg/kg i.v. and 5 and 20 mg/kg gavage doses
provided detectable plasma concentrations. GST-zeta depletion slowed DCA elimination
from plasma allowing kinetic analysis of doses as low as 0.05 mg/kg. DCA elimination
was strongly dose-dependent in the naive rats with total body clearance declining
with increasing dose. In the GST-zeta depleted rats, the toxicokinetics became
linear at doses 
1 mg/kg. Virtually all of the dose was eliminated through metabolic clearance
as the rate of urinary elimination was < 1 ml h-1.
At higher oral doses (
5 mg/kg in GST-zeta depleted and 20 mg/kg in naive), secondary
peaks in the plasma concentration appeared long after the completion of the
initial absorption phase. Although we do not know the specific mechanism causing
the formation of the secondary peaks after oral dosing, it is not associated
with enterohepatic circulation, as our earlier study established that DCA does
not undergo extensive biliary secretion (Schultz, et al., 1999). We hypothesized
that DCA absorption is region-dependent characterized by rapid absorption from
the stomach and/or upper regions of the of small intestine (i.e., duodenum and/or
jejunum), reduced absorption from the rest of the upper GI tract, which then
increases in the lower GI tract, and caused the secondary or multiple plasma
maxima. The specific mechanism that would reduce absorption in the upper GI
tract is unknown at present, but warrants further investigation because of the
potential importance in controlling DCA absorption and bioavailability. Oral
bioavailability of DCA was 0-13 percent in naïve and 14-75 percent in GST-zeta
depleted rats. Oral bioavailability of DCA to humans through consumption of
drinking water was predicted to be very low and less than 1 percent. The significance
of this study was the conclusion that use of the GST-zeta depleted rat model
for assessing the kinetics of DCA in humans can be justified by the similarity
in toxicokinetic parameter estimates and rate of in vitro metabolism of DCA
by human and GST-zeta depleted rat liver cytosol.
An additional aspect of GST-zeta metabolism discovered by this project (Schultz
and Sylvestor 2001), is the stereospecific nature of the reaction catalyzed
by this enzyme with chiral substrates. We studied the stereospecific toxicokinetics
of two chiral di-haloacetates in male F344 rats: (-), (+) bromochloro-acetate
(BCA), and racemic chlorofluoro-acetate (CFA), a non GST-zeta inhibiting di-haloacetate.
These experiments were repeated in animals that previously had been treated
with dichloroacetate (DCA) to deplete GST-zeta activity. Results indicated that
in naïve rats, the elimination half-life of (-)BCA was 0.07 hours as compared
to 0.40 hours for (+)BCA. A comparable difference in elimination half-life also
was observed for the CFA stereoisomers (0.79 hours versus 0.11 hours). In GST-zeta
depleted rats, stereospecific elimination of (-), (+) BCA was absent with both
stereoisomers, having an elimination half-life of approximately 0.4 hours. This
finding was in contrast to results for CFA, which still maintained the same
relative difference in elimination rate between its stereoisomers, although
overall elimination was diminished in GST-zeta depleted rats. The AUC0
of
(-), (+) BCA after oral administration differed by more than 10-fold in naïve
rats, but reduced considerably in GST-zeta depleted rats. The oral bioavailability
varied between 20-67 percent for the different BCA stereoisomers. The significance
of these results are that (+) BCA is a poor substrate for GST-zeta and the internal
dosimetry of (-), (+) BCA will differ considerably after drinking water exposure.
With regard to tri-HAs, nonlinear kinetics also has been observed. In a study reported by Merdink, et al. (2001), we examined the toxicokinetics of bromodichloroacetate (BDCA) in male B6C3F1 mice after oral and i.v. doses. BDCA was administered at a dose of 5, 20, or 100 mg kg-1 to the mice. The apparent volume of distribution (Vss) was similar to that observed in F344 rats (e.g., distributes within total body water). BDCA was subject to first-pass hepatic metabolism that was dose dependent, causing the oral bioavailabilities to vary from 0.28-0.73. A mean terminal half-life of 1.37 ± 0.21 h. was calculated from the two lower doses of both i.v. and oral administration. 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 to 2.3 h at the highest dose. The average CLb was 220 ml h-1 kg-1 for the lower two doses, but decreased to 156 mL h-1 kg-1 at the high dose. These results indicated that BDCA is primarily eliminated by metabolism, with only 2.4 percent of the parent dose recovered in urine after the high dose. The unbound renal clearance, as calculated from the high dose, was only 15.0 mL h-1 kg-1, only a small fraction of the total clearance. BDCA was moderately bound to plasma proteins with a blood/plasma ratio of 0.88, very similar to that reported in F344 rats. This study indicated that distribution of brominated tri-HAs is similar to rats, implying there is little species differences in Vss. The observations of high non-renal clearance, plus evidence for saturable elimination especially at doses above 20 mg/kg, indicated metabolism was important in determining both elimination and oral bioavailability.
These studies emphasized the complex differences between di- and tri-HAs regarding biotransformation. Although prolonged exposure to the tri-HAs does not appear to alter their rate of initial metabolism, the bromine substituted analogues undergo complex reactions in vitro, which appears to involve both cytosolic and microsomal pathways. In a study reported by Merdink, et al. (2000), experiments using bromo-dichloroacetate (BDCA) provided unambiguous proof that reductive de-bromination occurs in rodent microsomes. This was accomplished by trapping of the dichloroacetate radical intermediate using a spin-trapping agent phenyl t-butyl nitrone (PBN), and its subsequent identification by gas chromatography/mass spectrometry (GC/MS). This previously had been difficult to establish because the PBN/dichloroacetate radical adduct underwent an intramolecular rearrangement, which prevented characterization with electron paramagnetic resonance (EPR) techniques. We hypothesized that an internal condensation reaction between the acetate and the nitroxide radical moieties forms a cyclic adduct with the elimination of an OH radical and loss of the EPR signal. This would explain why previous studies of TCA failed to detect reductive dehalogenation reactions.
In summary, the significant finding from this study was that reductive dehalogenation does occur with tri-HAs and appears to proceed at a faster rate with bromine substituted analogues. We further characterized the metabolism of BDCA and two other brominated tri-HAs; chlorodibromo-acetate (CDBA) and tribromo-acetate (TBA), using liver microsomes from adult male Fischer 344 rats and specific P450 proteins. Reductive de-bromination was 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-HAs was 4-5 times higher under nitrogen headspace than under atmospheric conditions. Intrinsic metabolic clearance was of the order TBA>CDBA>>BDCA. At high substrate levels, the rate of consumption of the tri-HA was up to 2-3 times greater than the corresponding rate of formation of the di-HA metabolite (dichloro-, bromochloro-, or dibromo-acetate), 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 stoichometry). This result indicates the additional metabolite(s) formed does not release Br-. Carbon monoxide and diphenyleneiodonium (a specific P450 reductase inhibitor) blocked tri-HA metabolism. However, inhibitors of specific P450 proteins (CYP 2E1, 2D6, and 3A4) failed to significantly block metabolism. Metabolism of CDBA by CYP 2E1 and a mixture of six cytochrome P450s (CYP 1A2, CYP 2C8, CYP 2C9, CYP 2C19, CYP 2D6, and CYP 3A4) was 3-4 fold lower than rat liver microsomes, but comparable to rat cytochrome c reductase alone. These results indicate that at least two separate reactions were occurring: reductive dehalogenation leading to di-HA formation, and a second reaction that does not involve loss of a halogen.
In the final part of this project, a PBPK was developed for HAs that incorporates important features of HA kinetics (discussed above), such as the nonlinear elimination, and the complex plasma-time profiles observed for di-HAs after oral dosing, with secondary peaks appearing well after the initial absorption phase. We proceeded with development of the model in two phases. Our initial model was constructed for DCA and includes a multicompartment description of the gastrointestinal system to account for the observed discontinuous absorption of DCA (Stenner and Schultz, 2002). The model also describes liver metabolism using the dispersion (as opposed to the "well-stirred") model that is better able to account for the saturable metabolism and resultant nonlinearity in DCA elimination. Model predictions were validated against previously characterized plasma-time profiles for DCA and selected tissue concentration-time profiles obtained for this study. The second phase of model development describes the tissue levels of mixtures of di- and tri-HAs. To validate the model, we measured the tissue distribution, in vivo plasma/tissue partitioning, and oral bioavailability of two separate HA mixtures (DCA, CDBA, TBA; DBA, BCA, BDCA, and TCA) containing both di- and tri-HAs in F344 rats before and after depleting GST-zeta activity (Saghir and Schultz, 2002). Results indicated the plasma/tissue partition coefficients ranged between 0.6-1 and were similar to that observed from single HA dosing studies. The oral bioavailability of tri-HAs was not affected by GST-zeta depletion and ranged between 67-92 percent. The concentration of the HAs in selected tissues (liver, lung, muscle, kidney, testes, stomach, intestine) was similar to plasma concentrations as expected from the partition coefficients.
Journal Articles on this Report: 6 Displayed | Download in RIS Format
| Other project views: | All 29 publications | 9 publications in selected types | All 9 journal articles |
| Type | Citation | ||
|---|---|---|---|
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Arbuckle TE, et al. Assessing exposure in epidemiologic studies to disinfection by-products in drinking water: report from an international workshop. Environmental Health Perspectives. 2002;110 (Suppl 1):53-60 |
R825954 (Final) |
not available |
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Gonzalez-Leon A, Merdink JL, Bull RJ, Schultz IR. Effect of pre-treatment with dichloroacetic or trichloroacetic acid in drinking water on the pharmacokinetics of a subsequent challenge dose in B6C3F1 mice. Chemico-Biological Interactions 1999;123(3):239-253. |
R825954 (1999) R825954 (Final) |
not available |
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Saghir SA, Schultz IR. Low-dose pharmacokinetics and oral bioavailability of dichloroacetate in naive and GST zeta-depleted rats. Environmental Health Perspectives 2002;110(8):757-763 |
R825954 (Final) |
not available |
|
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Schultz IR, Merdink JL, Gonzalez-Leon AG, Bull RJ. Comparative toxicokinetics of chlorinated and brominated haloacetates in F344 rats. Toxicology and Applied Pharmacology 1999;158:103-114. |
R825954 (1999) R825954 (Final) |
not available |
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Schultz IR, Sylvestor S. Stereospecific toxicokinetics of bromochloro- and chlorofluoroacetate: Effect of GST-zeta depletion. Toxicology and Applied Pharmaoclogy 2001;175(2):104-113. |
R825954 (Final) |
not available |
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Schultz IR, Merdink JL, Gonzalez-Leon AG, Bull RJ. Dichloroacetate toxicokinetics and disruption of tyrosine catabolism in B6C3F1 mice: dose response relationships and age as a modifying factor. Toxicology 2002;173(3):229-247. |
R825954 (Final) |
not available |
drinking water, bioavailability, metabolism, enzymes, mixtures, halogenated acetic acids, toxicology, toxicokinetics, mass spectroscopy, tyrosine catabolism, microsomal, maleylacetoacetate. , Water, Scientific Discipline, RFA, Drinking Water, Analytical Chemistry, Health Risk Assessment, Environmental Chemistry, biomarkers, drinking water system, drinking water contaminants, treatment, haloacetic acids, exposure and effects, haloacids, metabolism, toxicokinetics, rodents, pharmacokinetics, tissue distribution, PBPK modeling, chemical byproducts, disinfection byproducts (DPBs), human health effects, DBP risk assessment, dose response, exposure, halogenated disinfection by-products
Progress and Final Reports:
1999 Progress Report
2000 Progress Report
Original Abstract