In Vivo Kinetics of Trichloroacetate in Male Fischer 344 Rats

Kyung O. Yu*, Hugh A. Barton{dagger}, Deirdre A. Mahle* and John M. Frazier*,1

* Operational Toxicology Branch, Human Effectiveness Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, Ohio 45433; and {dagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1
 APPENDIX 2
 APPENDIX 3
 REFERENCES
 
Trichloroacetate (TCA) is a toxicologically important metabolite of the industrial solvents trichloroethylene and tetrachloroethylene, and a by-product of the chlorination of drinking water. Tissue disposition and elimination of 14C-TCA were investigated in male Fischer 344 rats injected iv with 6.1, 61, or 306 µmol TCA/kg body weight. Blood and tissues were collected at various time points up to 24 h. No metabolites were observed in plasma, urine, or tissue extracts. Overall TCA kinetics in tissues were similar at all doses. Based on similar terminal elimination rate constants, tissues could be divided into three classes: plasma, RBC, muscle, and fat; kidney and skin; and liver, small intestine, and large intestine. Nonextractable radiolabel, assumed to be biologically incorporated metabolites in both liver and plasma, increased with time, peaking at 6–9 h postinjection. The fraction of the initial dose excreted in the urine at 24 h increased from 67% to 84% as the dose increased, whereas fecal excretion decreased from 7% to 4%. The cumulative elimination of TCA as CO2 at 24 h decreased from 12% to 8% of the total dose. Two important kinetic processes were identified: a) hepatic intracellular concentrations of TCA were significantly greater than free plasma concentrations, indicating concentrative transport at the hepatic sinusoidal plasma membrane, and b) TCA appears to be reabsorbed from urine postfiltration at the glomerulus, either in the renal tubules or in the bladder. These processes have an impact on the effective tissue dosimetry in liver and kidney and may play an important role in TCA toxicity.

Key Words: trichloroacetate; pharmacokinetics; in vivo; Fischer 344 rats; protein binding; elimination kinetics.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1
 APPENDIX 2
 APPENDIX 3
 REFERENCES
 
Trichloroethylene (TCE) has been used widely as a degreaser, dry cleaning solvent, spot remover, adhesive, lubricant, chemical intermediate, and anesthetic and analgesic agent (IARC, 1979Go). Due to its water solubility (0.1g/100 ml water) and extensive uses, TCE is a significant ground water contaminant (Bruckner et al., 1989Go; Kimbrough et al., 1985Go; Parchman and Magee, 1982Go; Waters et al., 1977Go). Trichloroacetic acid (TCA) is a major metabolite of TCE in rodents and human beings (Green and Prout, 1985Go; Odum et al., 1988Go). Water-soluble TCA is also a by-product of drinking water disinfection with chlorine (Jolly, 1985Go; Norwood et al., 1985Go; Uden and Miller, 1983Go). Therefore, human exposure to TCA is mainly from drinking water, either directly or as a metabolite of TCE.

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., 1987Go; Bull et al., 1990Go). Peroxisome proliferation by chloroacetic acids were reported in both mice and rats (DeAngelo et al., 1989Go; Elcombe, 1985Go; Odum et al., 1988Go). TCA is implicated in carcinogenicity and peroxisome proliferation in the liver (Bruckner et al., 1989Go). 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., 1989Go). 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, 1992Go; Templin et al., 1995Go). 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., 1988Go). Enterohepatic recirculation plays an important role in the kinetics of TCA formed via TCE metabolism (Green and Prout, 1985Go; Templin et al., 1995Go); however, TCA itself is not excreted in the bile in significant quantities (Toxopeus and Frazier, 1998Go). 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, 1980Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1
 APPENDIX 2
 APPENDIX 3
 REFERENCES
 
Chemicals.
Unlabeled trichloroacetic acid, acetonitrile, oxalic acid, dichloroacetic acid, glyoxalic acid, and glycolic acid were purchased from Sigma Chemical Co. (St. Louis, MO) and [1-14C] TCA (specific activity = 57 µCi/µmol) from American Radiolabeled Chemicals, Inc. (St. Louis, MO). HPLC analysis of radiolabeled TCA showed greater than 98% purity. Potassium hydroxide (85% pellets) was obtained from Aldrich Co. (St. Louis, MO). Carbo-Sorb E, PERMAFLOUR E+, and Pico-Fluor 40 were obtained from Packard Instrument Co. (Meriden, CT). All other chemicals used were of the highest grade commercially available.

Animals and treatment.
Male Fischer 344 rats weighing 195–235 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 (6–8 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 8–10 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 6–8 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1
 APPENDIX 2
 APPENDIX 3
 REFERENCES
 
The time course of the concentration of TCA (as TCA equivalents) in tissues after iv injection is presented in Figures 1A to 1FGo. The data reveal that overall kinetic behaviors are similar at all three doses (6.1, 61, and 306 µmoles/kg). At early time points, the highest concentration is observed in plasma, followed by kidney, RBC, liver, skin, small intestine, large intestine, muscle, and fat. This relative order of tissue concentrations is unchanged up to 3 h postdosing. TCA concentration in fat is significantly lower than other tissues at all dosing levels and time points. Plasma concentrations at 5 min postdosing with 6.1, 61, and 306 µmoles/kg are 29, 287, and 1080 µM, respectively. These concentrations scale roughly in proportion to the dose, although the concentration at the highest dose is less than what would be expected based on exact linearity (1080 versus 1450 µM). The terminal elimination rate constants are given in Table 1Go for all tissues and dosages. In general, disappearance of TCA equivalents from RBC, muscle, and fat is similar to or faster than plasma at all dosages. Kidney and skin have terminal disappearance rate constants that are consistently slightly lower than plasma, whereas liver, small intestine, and large intestine exhibit significantly slower elimination. In fact, the total concentration of TCA equivalents in liver exceeds that in plasma at 24 h postexposure. Possible explanations of this phenomenon are explored below.



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FIG. 1. Time course of plasma/tissue concentrations of TCA equivalents following an iv injection. (A) and (B): 6.1 µmoles of TCA/kg body weight; (C) and (D): 61 µmoles/kg body weight; (E) and (F): 306 µmoles/kg body weight. Data are means ± SD (n = 4).

 

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TABLE 1 Terminal First-order Rate Constants for TCA Disappearance from Plasma and Tissues
 
Urinary and fecal excretion of TCA are shown in Figure 2Go. Urinary excretion is rapid and dose dependent. Within 9 h postinjection, the fraction of the initial dose excreted in the urine at the low and high dosing levels is 43 ± 6% and 58 ± 5%, respectively; while at the end of 24 hr, cumulative urinary excretion is 67 ± 5% and 84 ± 6%. On the other hand, fecal excretion is low in general and at 24 h postexposure, the percentage of TCA equivalents eliminated at low and high doses is 7 ± 2% and 4 ± 1%, respectively. Total exhaled 14CO2 over 24 h at the low and high doses is 12 ± 1% and 8 ± 2%, respectively. The data indicate that the percentage of the dose eliminated in the urine increases at higher dosages, whereas that eliminated via the feces and exhaled in the breath decreases.



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FIG. 2. Cumulative urinary and fecal excretion of TCA. Total cumulative radiolabel derived from TCA excreted via urine (closed symbols) or feces (open symbols) following an iv injection is plotted (% of dose). Data are means ± SD (n = 4).

 
As noted above, the kinetics of TCA in the liver differ significantly from those in the plasma. One possible factor that could contribute to this behavior of TCA in the liver, relative to other tissues, is biologic incorporation of TCA metabolites into hepatic intracellular components. The total TCA radioactivity in a tissue can be experimentally resolved into extractable (ER) and nonextractable (NER) radiolabel and converted to concentrations of TCA equivalents. Such studies were conducted for plasma and liver at the lowest and highest doses, and the data are presented in Figures 3A and 3BGo. Limited data for the middle dose indicate a similar pattern (data not shown). In the plasma, the majority of the TCA equivalents were extractable at early time points. The concentration of NER increased up to 6 and 9 h postinjection (Fig. 3AGo) for the low and high dosages, respectively. After peaking, the NER remained relatively constant in the plasma (the t1/2 for clearance of NER in the plasma was significantly greater than 24 h). Hence, at the 24-h time point, the NER proportion in plasma increased to 21% and 50% of the total radiolabel at the lowest and highest dosages, respectively. The terminal elimination rate constant for the plasma ER component is consistently higher than that for the total plasma concentration at all doses (Table 1Go). Liver patterns (Fig. 3BGo) were similar, differing only in magnitude when compared to those in plasma. At 0.5 h postdosing, NER in liver was 8% and 17% at the low and high dosages, respectively. At the 24-h time point, 54% and 61% of the total radiolabel in the liver was accounted for by the NER component. Overall, NER was greater in the liver than in plasma on a concentration basis. The terminal elimination rate constant for the ER component is greater than that for the total TCA in liver, but is still consistently less than the terminal elimination rate constant for extractable TCA in plasma (Table 1Go).



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FIG. 3. Time course of total, extractable and non-extractable TCA equivalents in plasma (A) and liver (B). Data for the 6.1 µmole/kg dose (closed symbols) and the 306 µmoles/kg dose (open symbols) are plotted. Data are means ± SD (n = 4).

 
TCA is poorly metabolized in the rat. HPLC analyses of plasma, urine, and tissue homogenates were unable to detect any metabolites of TCA. The presence of 14CO2 indicates that some TCA is metabolized. In addition, the observation of nonextractable 14C-label in liver and plasma is thought to represent metabolites biologically incorporated into hepatic macromolecules that are either retained in the liver or secreted into plasma. Based on the amount of radiolabel either exhaled or incorporated into nonextractable components, the amount of TCA metabolized in the 24-h studies is less than 20% of the dose, and most of that occurs in the first 6–9 h after dosing.

From studies of the kinetics of TCA in the isolated perfused rat liver (Toxopeus and Frazier, 1998Go), 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 4BGo, respectively. Considerable binding of TCA to plasma is exhibited. The experimental plasma binding data were fitted to the following equation (Taira and Terada, 1985Go)

(1)
where CB (µM) is the bound concentration, CF (µM) is the free concentration, A (dimensionless) is a linear binding coefficient, BMAX (µM) is the maximum binding capacity of the saturable component, and KD (µM) is the dissociation constant. The estimated values for the binding parameters are A = 0.602, BMAX = 312 µM and KD = 136 µM. These data can be used to compute the free concentration of TCA in plasma, CP-FREE (µM), using the formula (see Appendix 1 for a derivation)

(2)
where CP-ER (µM) is the total concentration of extractable TCA in the plasma. The assumption is made that the nonextractable TCA equivalents in plasma are nonreversibly bound to plasma macromolecules and therefore are not in equilibrium with free TCA; only the extractable TCA equivalents are in equilibrium.The experimental determination of TCA binding to liver homogenate proved problematic. As a general rule, when binding is expressed as micromoles bound per millgram protein, the binding should be independent of dilution, i.e., as the sample is diluted, the binding at a given free concentration decreases, but so does the protein concentration in the same proportion; thus, the ratio remains constant. However, with liver homogenate, the binding per milligram protein was observed to increase with dilution. This could indicate the presence of a competitive inhibitor that is being diluted concurrently. Attempts to dialyze away a competitive inhibitor were unsuccessful, therefore this hypothesis could not be confirmed. Be that as it may, if the binding data are extrapolated to zero dilution, relatively little reversible binding of TCA to liver homogenates is observed. In the discussion that follows, the free concentration of TCA in the intracellular space of the liver is assumed to be approximately equal to the total concentration, i.e., binding of TCA in the intracellular space of the liver is assumed to be negligible. The experimental data are consistent with this assumption. Overall, the binding data indicate much stronger binding of TCA in plasma than in liver.Having obtained the binding parameters for TCA in plasma and demonstrated that the binding in liver is negligible, the next step is to calculate the total concentration of TCA in the liver intracellular space. A theoretical analysis of the distribution of a water-soluble chemical in a tissue (Appendix 2) can be used to derive an equation that will relate the concentration of extractable chemical in the intracellular water space to the total concentration of the chemical in the tissue after corrections for the presence of the chemical in the residual blood in the tissue. This equation is

(3)
where CiI-ER (µM) is the computed concentration of extractable chemical in the intracellular water space of the ith tissues, Ci-ER (µmoles/kg) is the total concentration of extractable chemical in the ith tissues, CiRBC-TOTAL (µmoles/kg) is the total concentration in the RBC, CiP-TOTAL (µM) is the total concentration in the plasma, CiE-TOTAL (µM) is the total concentration in the interstitial water space, HEME (dimensionless) is the hematocrit, FiV (L/kg) is the conversion factor to calculate the vascular volume from the tissue weight, FiE (L/kg) is the conversion factor to calculate the interstitial water volume, and FiI (l/kg) is the conversion factor to calculate the intracellular water volume. In general, the usefulness of this equation is limited for many tissues, as the concentration of the chemical in the interstitial space, CiE-TOTAL, is not usually measurable. However, this equation can be used to calculate the total concentration of TCA in the liver intracellular space at each sample time because the concentration of TCA in the hepatic interstitial space (space of Disse) can be assumed to be equal to the concentration in the plasma in the liver, CLE-TOTAL = CLP-TOTAL. This assumption is valid for the liver because small molecules in the plasma rapidly equilibrate with the space of Disse (Goreski et al., 1993Go).Equation 3Go gives the total concentration of the chemical in the intracellular water space of the liver. As discussed above, this is approximately equal to the free intracellular concentration, as intracellular binding can be neglected. In Figure 5Go, the calculated intracellular extractable TCA concentration, CLI-ER, is plotted as a function of the free plasma concentration for the highest TCA dose. Using the data from the highest dosage (306 µmol/kg), the intracellular concentration in the liver is observed to be greater than the free plasma concentration by a significant factor at all time points except the 5-min time point (corresponding to the data point at the highest free plasma concentration). The open symbols in Figure 5Go represent the data from the isolated perfused rat liver experiments (Toxopeus and Frazier, 1998Go). The in vivo data obtained from the high-dosage animals are consistent with the data obtained in the isolated perfused rat liver studies.Having corrected for nonextractable TCA and TCA binding, it is possible to compare the terminal rate constants for free TCA in plasma and free TCA in the liver intracellular space (as represented by CLI-ER; see Equation 3Go and related discussion). These rate constants are given in Table 1Go. The terminal elimination rate constants for liver intracellular TCA is still significantly less than the corresponding constants for free plasma TCA at all doses. This suggests that the transport processes that control the intracellular TCA concentration relative to the free plasma concentration are nonlinear. This nonlinear effect can be illustrated by plotting the ratio of the intracellular extractable TCA concentration to the free plasma concentration as a function of the free plasma concentration (Fig. 6Go). If it is assumed that the observed effect is due to asymmetrical transport, i.e., transport into liver cells per unit free concentration in the plasma is greater than transport out per unit free intracellular concentration, then an empirical equation of the form

(4)
can be used to fit the data (see Appendix 3 for a derivation). Fitting the experimental data for the high TCA dosage, gives {alpha} = 143 µM and ß = 15.4 µM. The fitted data are plotted in Figure 6Go. The mathematical relationship given by Equation 4Go is consistent with the experimental data. If this approach has any meaning, then the fitted parameter {alpha} is the ratio of the maximum saturable transport rate to the linear transport rate constant, while ß is equal to the free concentration at half-maximum transport via the saturable transport mechanism. It can also be shown that {alpha}/ß is the ratio of the saturable transport rate to the linear transport rate at free concentrations well below ß. Thus, at low free concentrations (well below 15.4 µM) the saturable transport rate is almost 10 times the linear transport rate. At high free concentrations, when the saturable transport mechanism is saturated, then the linear transport mechanism dominates.



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FIG. 4. Binding of TCA to rat plasma. (A): binding curve. (B): Scatchard plot. The solid line is a plot of the fitted binding curve of the form

where CB (µM) is the bound concentration, CF (µM) is the free concentration, A (dimensionless) is a linear binding coefficient, BMAX (µM) is the maximum binding capacity of the saturable component, and KD (µM) is the dissociation constant. A = 0.602, BMAX = 312 µM, and KD = 136 µM.

 


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FIG. 5. Comparison of total intracellular extractable TCA equivalents in liver to free TCA concentration in plasma. Total intracellular extractable TCA equivalents were computed using Equation 3Go in the text. In vivo data for the 306 µmole/kg dose are plotted (solid symbols) and data from isolated perfused rat liver studies (open symbols; Toxopeus and Frazier, 1998) are included for comparison.

 


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FIG. 6. Ratio of liver intracellular TCA concentration to free plasma TCA concentration. The hepatic intracellular TCA concentration was computed using Equation 3Go in the text and the free plasma TCA concentration was computed using Equation 2Go. The ratio data plotted is for the 306 µmole/kg dose. The solid curve is a plot of the fitted curve of the form

where {alpha} = 143 µM and ß = 15.4 µM.

 
Returning to the issue of urinary excretion of TCA, it is interesting to ask if the disappearance of TCA from the plasma is consistent with simple glomerular filtration at the kidney. This can be determined by computing the cumulative urinary excretion, assuming that free TCA in the plasma is filtered at the glomerulus and that no chemical is either gained or lost from the urinary tract following filtration. Fitting the free plasma concentration of TCA with multiple exponential terms provides the input to a simple model that integrates the rate of TCA filtration at the glomerulus to give the cumulative amount of TCA filtered. When this calculation is made, it is found that several times the total TCA dose is filtered at the glomerulus for all three doses investigated. This observation implies that significant reabsorption of TCA must be occurring from the urinary tract, either in the kidney or subsequently in the bladder.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1
 APPENDIX 2
 APPENDIX 3
 REFERENCES
 
One important component of risk assessment is a proper evaluation of the dosimetry of the active form of the toxicant at the molecular target in the target tissue. For TCA, the liver and kidney are critical targets in various test species. The objective of this study was to establish a database for TCA kinetics in the rat following iv injection in order to develop better quantitative estimates of target dosimetry. TCA is a highly water-soluble chemical with a low water–octanol partition coefficient. The kinetic data were collected with the idea of obtaining better information of TCA kinetics in relevant water compartments in the animal.

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. 4Go). 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 (67–84% of parent chemical was eliminated in the urine within 24 h). Larson and Bull (1992) reported that 48–65% 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, 1985Go). 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, 1985Go; Styles et al., 1991Go). 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 6.5–7.8% and was dose independent (Larson and Bull, 1992Go), 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., 1991Go). 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.


    APPENDIX 1
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1
 APPENDIX 2
 APPENDIX 3
 REFERENCES
 
Derivation of equation 2Go.
Empirically, it is assumed that binding consists of two components: a linear component and a saturable component. Then the bound concentration can be related to the free concentration by an equation of the form

(5)
where CB (µM) is the bound concentration, CF (µM) is the free concentration, A (dimensionless) is a linear binding coefficient, BMAX (µM) is the maximum binding capacity of the saturable component, and KD (µM) is the dissociation constant. The total concentration is the sum of the free and bound concentrations:

(6)

Substituting Equation A1-1Go for CB in Equation A1-2Go and solving for the free concentration using the quadratic formula gives

(7)


    APPENDIX 2
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1
 APPENDIX 2
 APPENDIX 3
 REFERENCES
 
Derivation of equation 3Go.
The total amount of a chemical in a tissue consists of the chemical contained in the plasma, RBC, and interstitial, and intracellular spaces. Thus, a mass balance equation can be written

(8)
where the subscript i refers to the ith tissue, Ci-ER (µmoles/kg) is the concentration of extractable TCA, Wi is the weight of the tissue (kg), CiP-TOTAL (µM) is the total concentration of TCA in the plasma, ViP (L) is the volume of plasma in the tissue, CiRBC-TOTAL is the total concentration of TCA in the RBC, ViRBC (L) is the volume of the RBC in the tissue, CiE-TOTAL (µM) is the total concentration of TCA in the interstitial space, ViE (L) is the volume of the interstitial space, CiI-TOTAL (µM) is the total concentration of TCA in the intracellular space, and ViI (L) is the volume of the intracellular space. This equation can be solved for CiI. Furthermore, the volumes of the various subcompartments of the ith tissue, Vij, can be expressed as

(9)
where Fij (L/kg) is the conversion factor that relates the volume of the subcompartment to the weight of the tissue (j = V for vasculature, = E for interstitial space, = I for intracellular space). In addition, the vascular space is divided into the plasma space and the RBC space based on the hematocrit of the blood that occupies the vascular space. Making these substitutions, the equation for the concentration of the chemical in the intracellular space becomes

(10)


    APPENDIX 3
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1
 APPENDIX 2
 APPENDIX 3
 REFERENCES
 
Derivation of equation 4Go.
It is assumed that transport into the hepatocyte is the sum of two components, a linear component and a saturable component, and that transport out is only a linear component. Then at quasi-steady state the rate of transport into the cell is approximately equal to the rate of transport out

(11)
where it is further assumed that the linear component of transport is symmetrical and is determined by the permeability P (cm/sec), and the saturable component can be represented by a Michaelis-Menton type relationship with a maximum transport rate of TMAX (nmol/sec * cm2) and a half-maximum concentration of KT (µM). The area of the interface between the plasma and the intracellular compartment is A (cm2). Note that the 3.6 factors are included to convert transport rate units to micromoles per hour. This equation can be solved for the ratio of CLI-FREE to CP-FREE. Thus,

(12)
which has the mathematical form of

(13)

As there is little intracellular binding of TCA in the liver, CLI-FREE {approx} CLI-ER. Substituting for CLI-FREE gives Equation 4Go.


    NOTES
 
1 To whom correspondence should be addressed at AFRL/HEST, Bldg. 79, 2856 G Street, Wright-Patterson Air Force Base, OH 45433-7400. Fax: (937) 255-1474. E-mail: john.frazier{at}he.wpafb.af.mil. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1
 APPENDIX 2
 APPENDIX 3
 REFERENCES
 
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