* Operational Toxicology Branch, Human Effectiveness Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, Ohio 45433; and
Pharmacokinetics Branch, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received September 24, 1999; accepted December 10, 1999
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ABSTRACT |
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Key Words: trichloroacetate; pharmacokinetics; in vivo; Fischer 344 rats; protein binding; elimination kinetics.
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INTRODUCTION |
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Toxicologic concerns about TCA center on the carcinogenic potential of the chemical. In drinking water studies, TCA produced liver tumors in B6C3F1 mice (Herren-Freund et al., 1987; Bull et al., 1990
). Peroxisome proliferation by chloroacetic acids were reported in both mice and rats (DeAngelo et al., 1989
; Elcombe, 1985
; Odum et al., 1988
). TCA is implicated in carcinogenicity and peroxisome proliferation in the liver (Bruckner et al., 1989
). In addition to the carcinogenic potential of TCA, teratogenic effects of TCA have been reported in rats, resulting in a dose-dependent reduction of weight and length of live fetuses, and soft tissue malformations, especially in the cardiovascular system (Smith et al., 1989
). To better understand these toxicologic effects, target tissue dosimetry can provide important insights into exposure-response relationships.
There are species-specific differences in TCA excretion after TCE exposure (Larson and Bull, 1992; Templin et al., 1995
). However, in all cases, urine is the major elimination route of TCA. Urinary excretion may be confounded by reabsorption in the bladder. It was observed that almost 70% of TCA injected into dog bladders was absorbed into the systemic circulation, suggesting that reabsorption from the bladder after glomerular filtration may play a role in TCA kinetics (Hobara et al., 1988
). Enterohepatic recirculation plays an important role in the kinetics of TCA formed via TCE metabolism (Green and Prout, 1985
; Templin et al., 1995
); however, TCA itself is not excreted in the bile in significant quantities (Toxopeus and Frazier, 1998
). Recently, Stenner et al. (1997) demonstrated that 76% of the TCA in blood following TCE exposure was derived from trichloroethanol, which is subject to enterohepatic recirculation as the glucuronide.
TCA has a pKa of 0.9 and is fully charged under physiologic condition (pH of ~7.4) and predominantly charged even in the stomach. Acidic compounds such as TCA, phenoxyacetic acids, and substituted benzoic acids bind with serum albumin (Fang and Lindstrom, 1980). Plasma binding plays an important role in the tissue distribution and elimination of TCA, as only free TCA is available to tissues for uptake and elimination.
The objective of this study was to investigate the dose dependency of TCA kinetics in the rat. Concentrations in plasma and eight tissues were determined over a 24-h period following iv injection at three dose levels to provide a complete kinetic profile. Urine, feces, and expired air were monitored to account for all routes of elimination. Particular attention was focused on reversible binding of TCA in plasma and liver. In addition, nonextractable radiolabel was determined to evaluate biologic incorporation of TCA metabolites into plasma and liver biomolecules. The data from these in vivo kinetic studies can be used to develop better estimates of target organ dosimetry.
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MATERIALS AND METHODS |
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Animals and treatment.
Male Fischer 344 rats weighing 195235 g were purchased from Charles River, Inc. (Raleigh, NC) and were randomly divided into 6 groups (n = 4). Animals were maintained on Purina Lab Chow and deionized water ad libitum. [1-14C] TCA was mixed with unlabeled TCA to prepare three dosing solutions with total concentrations of 6.1, 61, and 306 µmol TCA/ml in physiologic saline. Each animal received approximately the same amount of radiolabel (68 µCi of 14C per rat), thus the specific activity ranged from 13 to 0.2 x 106 dpm/µmol TCA for the lowest to highest dose, respectively. The specific activity was calibrated for each dosing solution. Rats were injected via the lateral tail vein with a volume of 1 ml/kg, giving doses of 6.1, 61, and 306 µmol/kg, and placed in metabolism cages. Rats intended for sacrifice at the 24-h time point were placed in glass metabolism cages equipped with airflow systems capable of scrubbing exhaled CO2 in a 100-ml trap filled with 2 M KOH. A flow of 500 ml/h of air was passed through the cage and the trap. Dosed rats were sacrificed via CO2 asphyxiation at 0.083 (5 min), 0.5 (30 min), 1, 3, 6, 9, and 24 h postinjection. Blood samples were drawn from the vena cava and centrifuged (3000 rpm x 10 min) to separate plasma and red blood cells (RBC); hematocrit was determined. Liver, kidney, muscle (lateral thigh), skin (lateral thigh, unshaven), fat (peritoneal), small intestine, and large intestine were removed and stored at 20°C until analyzed. Note: small and large intestine samples were not collected for the 61 µmole/kg dose. Feces and urine were collected at 3-, 6-, 9-, and 24-h time points. Triplicate samples of tissues (100 ± 20 mg each) were oxidized in a Packard Oxidizer (Model 304). 14CO2 was collected in Carbo-Sorb E and Permaflour E+, and radioactivity was determined by liquid scintillation counting using a Packard liquid scintillation analyzer (Tri-Carb 2200CA). Aliquots of urine (0.1 ml) and exhaled breath trapped in the 2 M KOH solution (1.0 ml) were mixed with 14.0 ml Pico-Fluor 40 and counted. All data derived from radiolabel analyses are expressed as TCA equivalents using specific activity. Animal use described in this study was conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996, and the Animal Welfare Act of 1966, as amended.
HPLC analysis.
One gram of liver was homogenized with an equal volume of deionized water. Aliquots (300 µl) of liver homogenate, plasma or urine were transferred to test tubes, and 600 µl of ice-cold acetonitrile was added to each tube. The tubes were vortexed for 810 s and incubated on ice for 1 h. Samples were centrifuged at 2500 rpm for 15 min at 4°C. The acetonitrile layer was drawn off, filtered into a test tube, and evaporated under nitrogen until about 200 µl of liquid remained. Sample volumes were brought back to 300 µl with deionized water and filtered into HPLC microvials. Parent TCA was separated from its potential metabolites by HPLC using a Rezek organic acid column (300 x 7.8 mm; Phenomenex, Torrance, CA). Mobile phase and flow rate were 0.005N H2SO4 and 1 ml/min, respectively. Cold TCA and metabolites were detected using a Waters 994 Photodiode Array detector at 210 nm and radiolabeled compounds using a Packard Radiomatic Detector (Model 280). Ultima-Flo M scintillation cocktail was employed at a ratio of 3:1 (cocktail to mobile phase). TCA and its potential metabolites were eluted as follows: TCA, 10.7 min; oxalate, 11.5 min; dichloroacetate, 14.9 min; glyoxalate, 15.4 min; and glycolate, 19.4 min.
Extractable and nonextractable TCA.
Aliquots (300 µl) of liver homogenate (1 g liver to 3 ml deionized water) or plasma were added to glass test tubes. Two volumes of ice-cold acetonitrile were added to each tube and vortexed for 68 s. The samples were incubated on ice for 1 h, then centrifuged at 1000 rpm for 5 min to separate liquid and solid phases. Low-speed centrifugation was used to avoid packing the precipitate. Five hundred microliters of the acetonitrile layer were drawn off, mixed with 20 ml Pico-Fluor 40 and quantitated by liquid scintillation counting. The precipitate was washed four times with 500 µl acetonitrile. Each time, the supernatant was collected and analyzed by liquid scintillation counting. The total TCA equivalents in the sum of all the extractions was considered the extractable TCA. The precipitate was dried and combusted using a Packard oxidizer (Model 304). The 14CO2 trapped in Carbo-Sorb and Permafluor E+ was determined by liquid scintillation techniques as described above. The total TCA equivalents in the precipitate were considered the nonextractable TCA. Note: the amount of radioactivity in these fractions are reported as TCA equivalents, taking into consideration that the radioactivity may no longer be associated with the parent chemical.
TCA binding studies.
Male F-344 rats were sacrificed via CO2 asphyxiation, and blood was drawn via the inferior vena cava. Plasma was separated from RBC by centrifugation at 3000 x g for 15 min. Plasma was used without dilution. The liver was perfused with heparinized Tris-KCl buffer (0.05 M Tris, 0.154 M KCl) to remove residual blood, and homogenized in Tris-KCl buffer (1 g tissue to 3 ml Tris-KCl buffer). The homogenate was frozen at 80°C until analysis. Protein concentrations were determined for plasma and liver homogenate by the BCA microtiter plate method (BCA Protein Assay Kit No. 23225, Pierce, Rockford, IL). During preparation of homogenates, all glassware and reagents were maintained at 4°C to minimize enzymatic degradation.
The binding of [14C]TCA in plasma and liver homogenate was investigated. The procedures used were the same as reported in Templin et al. (1995). Briefly, plasma or liver homogenate (390 µl) was placed into 2-ml amber vials, and an aliquot of [14C]TCA (10 µl) was added. Ten concentrations varying from 3.8 to 1530 µM TCA were investigated. The mixtures were incubated at 37°C for a period of time that was optimized for each tissue. At the end of the incubation period, 40 µl of the incubate were mixed with Pico-Fluor 40 (14 ml) and the total radioactivity was determined by liquid scintillation counting. Radioactivity (dpm) was converted to TCA equivalents; the concentration obtained represents the total TCA concentration. The remainder of the incubate was place into a Centrifree micopartition device (Amicon, Inc., Beverly, MA), and centrifuged at 2000 x g until sufficient ultrafiltrate was obtained for analysis. An aliquot of ultrafiltrate (40 µl) was mixed with Pico-Fluor 40 (14 ml) and assayed for radiolabel. The TCA equivalent concentration in the ultrafiltrate represents the free TCA concentration. Standard binding curves and Scatchard analysis were used to evaluate TCA binding parameters.
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RESULTS |
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From studies of the kinetics of TCA in the isolated perfused rat liver (Toxopeus and Frazier, 1998), the possibility that the free concentration of TCA in the liver intracellular space may be elevated relative to that in the perfusing medium was suggested. To investigate whether this possibility occurs in vivo, it is necessary to compute the free concentration of TCA in the plasma and compare it to the calculated free concentration of TCA in the intracellular space of the liver. The first step in making these calculations is to determine the binding parameters for TCA in rat plasma and in liver homogenates.
To determine the TCA-binding parameters, in vitro binding studies in plasma and liver homogenates were conducted. The binding curve and the Scatchard plot for TCA binding to rat plasma are shown in Figures 4A and 4B, respectively. Considerable binding of TCA to plasma is exhibited. The experimental plasma binding data were fitted to the following equation (Taira and Terada, 1985
)
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DISCUSSION |
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The overall kinetics of TCA in the rat are consistent with the behavior of a highly water-soluble, poorly metabolized chemical. The parent chemical is excreted mostly in the urine and the lowest tissue concentration is found in the fat tissue. The initial distribution of TCA in tissues is relatively independent of dose, although some slight nonlinear behavior is noted at the highest dose. The most prominent nonlinearity is the dose dependency of the cumulative urinary excretion. This effect is consistent with the nonlinear nature of the binding of TCA to plasma proteins (Fig. 4). At the higher doses, a greater portion of the TCA in plasma is free due to partial saturation of plasma binding. Thus, proportionately more TCA is available for glomerular filtration and urinary excretion at early time points. This is reflected in the greater fraction of TCA excreted in urine in the first 6 h at the highest dose.
The systemic clearance of TCA after iv dosing is dominated by the urinary excretion of unchanged TCA (6784% of parent chemical was eliminated in the urine within 24 h). Larson and Bull (1992) reported that 4865% of the initial doses was eliminated in urine following oral doses of 5, 20, and 100 mg TCA/kg to F-344 rats. However, only 35% of a 10-mg/kg TCA dose was excreted in the first 24 h after iv injection of Osbone-Mendel rats (Green and Prout, 1985). Differences in both dosing route and strains of rat may have contributed to different patterns in TCA elimination kinetics. CO2 has been shown to be a major metabolite of TCA in rats and mice (Green and Prout, 1985
; Styles et al., 1991
). The percentage of total elimination (12%) at the low dose studied here is comparable to that described by Green and Prout (1985), who reported that 12% of the total dose was eliminated as CO2 after iv injection of TCA. However, different kinetic behaviors were obtained when a wide range of TCA was orally administered to Fischer 344 rats. At 48 h postdosing, the percentages of radiolabel eliminated as CO2 was 6.57.8% and was dose independent (Larson and Bull, 1992
), whereas our study showed a slight dose dependency. Again, route of exposure may be an important determinant of kinetic pathways.
TCA is poorly metabolized in the rat. No DCA or other metabolites were found in plasma, urine, or tissues in our study. This result is consistent with that reported by Templin et al. (1995). However, Larson and Bull (1992) identified glyoxylate, oxalate, glycolate, and a small amount of DCA in urine following oral dosing with TCA.
The kinetic behavior of TCA in the liver initially attracted our attention, as it appeared to be inconsistent with the plasma kinetics. The studies to evaluate the nonextractable component of the TCA in plasma and liver were conducted to resolve this issue. Previous studies in the literature have demonstrated that [14C] derived from labeled TCA was bound to hepatic macromolecules in mice (Styles et al., 1991). These investigators reported that the percentage of liver binding increased from 3% at 1 h to 43% at 24 h in the mouse liver. Our data for Fischer 344 rats indicated an increase from 10% to 60% in the rat liver over the same period of time. Thus, both rats and mice show a time-related increase of TCA-derived radioactivity bound to hepatic macromolecules.
The present study demonstrated that there was dose- and time-dependent increase in incorporation of radiolabel into the nonextractable pool in plasma. Styles et al. (1991) found little covalently bound radioactivity in plasma after oral dosing with TCA in mice. Whether this discrepancy is a species effect or a route effect is not clear. Stevens et al. (1992) investigated the covalent binding of TCA-derived radioactivity in albumin and hemoglobin in both rats and mice. Radiolabel was shown to be incorporated into both proteins in both species. At the present time, it is not possible to determine whether TCA or its metabolite(s) binds covalently with macromolecules or whether radiolabel derived from TCA is metabolically incorporated into macromolecules. Further investigations are needed to clarify the issue.
Having corrected the plasma and liver data for nonextractable and bound TCA, the kinetics of TCA in the two tissues are still not consistent. TCA leaves the liver much more slowly than it disappears from the plasma. It is hypothesized that this effect is a consequence of saturable asymmetric transport of TCA across the hepatocyte plasma membrane. The experimental data are consistent with this hypothesis; however, additional studies with membrane vesicles prepared from rat liver would be useful to test this hypothesis.
The theoretical evaluation of the cumulative urinary excretion of TCA, assuming glomerular filtration of free TCA from the plasma is the only process operable, indicated that TCA should be eliminated in the urine much more rapidly than is observed. It was suggested that reabsorption in the renal tubules and/or from the bladder may be occurring. As was mentioned in the introduction, Hobara et al. (1988) observed reabsorption of TCA into the systemic circulation after injection into the dog bladder. Thus, the possibility of reabsorption from the bladder cannot be ruled out. A reabsorption mechanism in the kidney could also be important and could account for the high concentration of TCA in the kidney relative to other tissues. The consequence of postglomerular reabsorption is that TCA is retained in the systemic circulation for a longer period of time than would be the case if reabsorption did not occur.
In conclusion, we report the time course of tissue disposition of TCA in the rat at three doses following iv injection. Similar kinetic behavior is observed at all three dose levels. Limited metabolism of TCA occurs, and urinary elimination of the parent chemical is the major route of elimination. Binding of TCA to plasma proteins is a controlling factor in the systemic kinetics of TCA. A nonextractable component of plasma and liver TCA is detected and this component becomes dominant at later time points. TCA appears to be concentrated in the liver intracellular space relative to the free concentration in the plasma, and TCA filtered at the glomerulus appears to be reabsorbed from the urinary tract. These factors are potentially important in target dosimetry of TCA and could be relevant to species differences in TCA toxicity.
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APPENDIX 1 |
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Substituting Equation A1-1 for CB in Equation A1-2
and solving for the free concentration using the quadratic formula gives
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APPENDIX 2 |
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APPENDIX 3 |
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As there is little intracellular binding of TCA in the liver, CLI-FREE CLI-ER. Substituting for CLI-FREE gives Equation 4
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NOTES |
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REFERENCES |
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