* Research Institute of Toxicology, Utrecht University, P.O. Box 80176, 3508-TD Utrecht, The Netherlands; and
Leiden Advanced Pharmacokinetics & Pharmacodynamics (LAP&P) Consultants, Archimedesweg 31, 2333 CM Leiden, The Netherlands
Received January 9, 2001; accepted June 1, 2001
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ABSTRACT |
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Key Words: PB-PK; PCB; rat; in vitro-in vivo extrapolation; partition coefficients; metabolism; toxicokinetics.
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INTRODUCTION |
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Physiologically based pharmacokinetic (PB-PK) models have been used successfully to analyze the pharmacokinetic behavior of lipophilic substances like PCBs and dioxins (Andersen et al., 1993; Kedderis et al., 1993
; Lutz et al., 1977
, 1984
). The parameters of such models may be obtained a priori, either from literature, in vitro studies, or by fitting the model to the experimental data or from in vivo experiments (Yang and Andersen, 1994
). Extrapolation of in vitro metabolic rates resulted in good estimates for the in vivo metabolism of volatile and nonvolatile organic chemicals (Carlile et al., 1998
; Houston, 1994
; Houston and Carlile, 1997
; Krishnan and Andersen, 1994
), but lipophilic substances, like TCBTs have not been studied in this respect.
The aim of the present study was to determine the kinetics of TCBTs and to determine the potential of these compounds to accumulate in mammals. Hence, rats were intravenously injected with a single bolus dose of a mixture of 3 TCBT isomers (Fig. 1). The tissue concentrations were determined at various time points after dosing, and analyzed using a PB-PK model. Furthermore, in vitro metabolic rates were compared to in vivo metabolic rate constants.
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MATERIALS AND METHODS |
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Animals.
Adult male CPB:UwU rats (250300 g) obtained from the Central Animal Laboratory Utrecht University were acclimatized for 5 days. Rats were housed individually in Macrolon III cages with filter top. Animals were maintained on a 12-h light/dark cycle under conditions of constant temperature and humidity. The rats were provided with rodent chow and tap water ad libitum. After 5 days the rats were weighed and dosed intravenously with a mixture of TCBT 87 (0.43 mg/kg), TCBT 88 (0.45 mg/kg), and TCBT 94 (0.48 mg/kg). The mixture was dissolved in a 20% tween/water (v/v) solution (polyethylenesorbitanmonooleate, Sigma, St. Louis, MO) and administered via the tail vein at a constant volume of 1 ml/kg. In vitro studies demonstrated that no interactions with respect to metabolism (Kramer et al., 2000) occurred at the blood concentrations obtained after application of this dose.
Animals were killed in groups of 3 over a period of 168 h (t = 6, 10, 24, 48, 96, and 168 h) after sedation with CO2 (g), which was followed by exsanguination by aorta punction. Blood was collected in glass tubes with EDTA as anticoagulant and stored at 4°C until further use. About 1 to 4 g of liver, abdominal skin, abdominal adipose tissue, and muscle from the hind leg were collected. The tissues and organs were weighed and stored at 80°C until further use. Blood from the liver was removed by perfusing with saline.
Extraction of tissues.
The frozen tissues and organs (except for blood samples) were freeze dried for 48 h. The dried samples were placed in a paper cartridge and spiked with 100 µl of PCB 153 (1.6 mg/l; internal standard) in n-hexane. Subsequently the samples were extracted with 80 ml n-hexane under reflux for 16 h (Bouraly, 1989). The recovery of the extraction was 90 ± 5.6 %. The extract was collected in preweighed flasks and the n-hexane was evaporated under nitrogen. The n-hexane extractable fat was then determined gravimetrically.
Cleanup.
Glass columns (1 cm, 20 cm length) were filled with 4 grams Florisil (mesh 60200) deactivated with 5% water (w/w) to remove the n-hexane extractable fat (Bouraly, 1989). The column was pre-eluted with 5 ml n-hexane before adding 1 ml of the tissue sample containing maximally 100 mg fat extract. The samples were eluted with 30 ml n-hexane. The recovery of the cleanup over the column was 70 ± 3.7 %. The eluates were concentrated prior to gas chromatographic analysis to a volume of ca. 500 µl.
Blood sample cleanup.
Blood was extracted using 2 ml acetone and 2 ml n-hexane per gram blood (De Boer and Wester, 1993). Prior to the extraction PCB153 (1.6 mg/l) was added as an internal standard. The mixture was shaken for 30 s and centrifuged for 1 min at 3000 rpm. The extraction was repeated 3 times. The organic phase was collected in preweighed vials and evaporated at room temperature under nitrogen. The n-hexane:acetone extractable fat content was determined gravimetrically. The recovery was 101 ± 10%.
GC analysis.
The extracts were analyzed on a Carlo Erba Mega 5360 HR-GC-63Ni-Electron Capture Detector equipped with a 15 m DB5 column (ID 0.32 mm, film thickness 0.25 µm). Samples were analyzed using splitless injection according to the following temperature program. The analysis started at 70°C, after 2 min temperature increased to 255°C at 20°C/min temperature. After 5 min at 255°C, temperature increased to 280°C at a rate of 30°C/min. After 4 min at 280°C the analysis stopped. The injector temperature was set at 250°C. Splitless closing times were 30 s preinjection and 99 s postinjection. The injection volume was 2 µl. The peak areas of the different compounds were determined relative to the peak area of PCB 153. Calibration curves for each compound using the internal standard PCB153 (1.6 mg/l) were used to quantify the amount per sample. This amount was divided by the wet weight of the tissue or blood sample.
Model description.
The general model structure (Fig. 2) was based on a model for PCBs (Lutz et al., 1977
, 1984
).
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Model parameterization.
The physiological parameters are presented in Table 1. The whole body volume has been taken into account except for the bone tissue (8%) because of its limited perfusion (Brown et al., 1994
). Tissue to blood partition coefficients (PC) were calculated according to Gallo et al. (1987). The PC for remaining tissue was set equal to the value for muscle (Table 2
).
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Simulation software.
The model equations (Appendix) were implemented in ACSL for Windows (version 11.4.1., MGA Software Inc., Concord, MA). Nelder-Mead (simplex) algorithm was used to optimize the model parameters in ACSL math (version 1.2, MGA Software Inc.).
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RESULTS |
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The metabolic rate constant of TCBT 87 was about 5 times lower than the metabolic rates of TCBT 88 and 94. The precision of the estimation of the metabolic rates, as indicated by the coefficient of variation (CV), ranged from 610%, and was comparable among the isomers. The diffusional clearance constants for TCBT 87 in each tissue tended to be higher than for the other 2 compounds. These estimates were less precise relative to the metabolic rate constants for all 3 isomers, with CVs ranging from 0.5 to 35%.
In vitro metabolic rate constants, as previously determined in hepatic microsomes from male rat (Kramer et al., 2000) were compared to in vivo metabolic rate constants (Table 3
). The in vitro values were a factor 2 to 3 smaller than the metabolic rate constants, obtained after optimizing the model to the in vivo data. Alternatively, optimizing the diffusional clearance only, while fixing the metabolic rate to the in vitro values, overestimates systematically the tissue and blood concentrations (data not shown).
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DISCUSSION |
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Although the octanol to water partition coefficients of the TCBTs lie in the same range (log Kow 7.37.4), large differences in partition coefficients between the individual isomers were presently observed. TCBT 87 appeared to have the largest PCs for all tissues and the lowest fat to liver concentration ratio. The partitioning of TCBT 87 to the liver is relatively high (Pl = 25), compared to Pls used in PB-PK models for related compounds. These Pls ranged from 3 to 17 (Kedderis et al., 1993; Lutz et al., 1977
; Tuey and Matthews, 1980
). TCDD and related compounds, like some PCBs, are selectively retained by CYP1A2 in the liver, causing alterations in the fat to liver concentration ratios, comparable to that observed for TCBT 87 (Andersen et al., 1997
, 1993
; DeVito et al., 1998
; Van den Berg et al., 1994
). This suggests that the differences in tissue partitioning among the TCBTs could be explained by selective retention of TCBT 87 by CYP1A2 in the liver.
TCBT 87 also differs from the other 2 isomers with respect to its metabolic rate. TCBT 87 was metabolized slower than TCBT 88 and TCBT 94. This agrees with the results of the in vitro study (Kramer et al., 2000). The present study showed that in vitro metabolic rate constants were 2- to 3-fold lower than the estimated in vivo metabolic rate constants. This could be explained by the possible contribution of other tissues to the metabolism in vivo, since the in vitro data were based on metabolism in the liver only. Another aspect could be the simultaneous estimation, which could confound the values. Since the correlation matrix did not reveal significant correlations, this effect was considered limited. However, differences in binding characteristics between in vitro and in vivo may be more likely, as was demonstrated for various drugs by Obach (1997). In this study, the extrapolation of in vitro data from experiments with microsomal fractions was improved when the free fractions of the substrate in the in vitro system and in vivo were taken into account. Thus, the factor of 2 to 3 between in vitro and in vivo might reflect the differences in the free fraction.
The direct insertion of these in vitro values in the PB-PK model resulted in an overestimation of the tissue concentrations.
Sensitivity of the metabolic rate constant on the model predictions of the amount metabolized showed that this parameter played an important role during the distribution phase of the TCBTs. This observation was substantiated by the almost similar terminal half-lives of the TCBTs, while the in vivo metabolic rate constants of the TCBTs differed maximally by a factor of 5 (Table 3). The blood flow through the liver also contributed substantially to the elimination as indicated by the normalized sensitivity coefficient (Fig. 4
). These findings suggest that processes other than the metabolism become rate limiting during the terminal elimination phase, e.g., redistribution from the adipose tissue.
In summary, the in vivo kinetics of TCBTs were comparable to that of PCBs, with the highest tissue concentrations in the adipose tissue. Furthermore, it was demonstrated that the tissue concentration data were adequately described by the PB-PK model. This model showed that TCBT 87 was distinct from the other 2 TCBTs with respect to tissue partitioning and metabolic rates. Additionally, the model illustrated that during the distribution phase, metabolism played an important role in the elimination of the TCBTs, while during the terminal elimination phase possible redistribution from tissue to blood becomes rate limiting.
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APPENDIX |
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![]() | (1) |
![]() | (2) |
The amount in kidney and remaining tissue are described as follows:
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Concentrations in these tissues are represented by the amount divided by the tissue volume:
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Metabolism is described as a first-order process, which occurs in the liver. The liver compartment is therefore described as:
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where Kmet represents the metabolic rate constant.
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where Qx is the blood flow through tissue x and PAxC is a constant representing part of the diffusional clearance (PAx).
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NOTES |
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2 To whom correspondence should be addressed at Gebouw Nieuw Gildestein, Yalelaan 2, 3584 CL Utrecht, The Netherlands. E-mail: w.seinen{at}iras.uu.nl.
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REFERENCES |
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Andersen, M. E., Mills, J. J., Gargas, M. L., Kedderis, L., Birnbaum, L. S., Neubert, D., and Greenlee, W. F. (1993). Modeling receptor-mediated processes with dioxin: Implications for pharmacokinetics and risk assessment. Risk Anal. 13, 2536.[ISI][Medline]
Bouraly, M., and Millischer, R. J. (1989). Bioaccumulation and elimination of tetrachlorobenzyltoluene (TCBT) by the rat and by fish. Chemosphere 18, 20512063.[ISI]
Brown, R., Foran, J., Olin, S., and Robinson, D. (1994). Physiological Parameter Values for PBPK Models. International Life Sciences Institute and Risk Science Institute, Washington D. C.
Carlile, D. J., Stevens, A. J., Ashforth, E. I., Waghela, D., and Houston, J. B. (1998). In vivo clearance of ethoxycoumarin and its prediction from in vitro systems. Use of drug depletion and metabolite formation methods in hepatic microsomes and isolated hepatocytes. Drug Metab. Dispos. 26, 216221.
De Boer, J., and Wester, P. G. (1993). Determination of toxaphene in human milk from Nigaragua and in fish and marine mammals from the northeastern Atlantic and the North Sea. Chemosphere 27, 18791890.[ISI]
De Lang, R. -J., van Hooijdonk, M. J. C. M., Brandsma, L., Kramer, H., and Seinen, W. (1998). Transition metal catalysed cross-coupling between benzylic halides and aryl nucleophiles. Synthesis of some toxicologically interesting tetrachlorobenzyltoluenes. Tetrahedron 54, 29532966.[ISI]
DeVito, M. J., Ross, D. G., Dupuy, A. E., Jr., Ferrario, J., McDaniel, D., and Birnbaum, L. S. (1998). Dose-response relationships for disposition and hepatic sequestration of polyhalogenated dibenzo-p-dioxins, dibenzofurans, and biphenyls following subchronic treatment in mice. Toxicol. Sci. 46, 223234.[Abstract]
Ehmann, J., and Ballschmiter, K. (1989). Isomer-specific determination of tetrachlorobenzyltoluenes (TCBT) in the technical mixture Ugilec 141 by capillary gas chromatography. Fresenius Z. Anal. Chem. 332, 904911.
Evans, M. V., Crank, W. D., Yang, H. -M., and Simmons, J. E. (1994). Applications of sensitivity analysis to a physiologically based pharmacokinetic model for carbon tetrachloride in rats. Toxicol. Appl. Pharmacol. 128, 3644.[ISI][Medline]
Fuerst, P., Krueger, C., Meemken, H. -A. and Groebel, W. (1987). Levels of PCB-substitute Ugilec (tetrachlorobenzyltoluenes) in fish from areas with extensive mining. Z. Lebensm. Unters. Forsch. 185, 394397.[ISI][Medline]
Gallo, J. M., Lam, F. C., and Perrier, D. G. (1987). Area method for the estimation of partition coefficients for physiological pharmacokinetic models. J. Pharmacokinet. Biopharm. 15, 271280.[ISI][Medline]
Heinrich-Hirsch, B., Beck, H., Chahoud, I., Grote, K., Hartmann, J., and Mathar, W. (1997). Tissue distribution, toxicokinetics and induction of hepatic drug metabolizing enzymes in male rats after a single s.c. dose of 3,4,3',4'-tetrachlorobiphenyl (PCB-77). Chemosphere 34, 15231534.[ISI][Medline]
Houston, J. B. (1994). Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem. Pharmacol. 47, 14691479.[ISI][Medline]
Houston, J. B., and Carlile, D. J. (1997). Prediction of hepatic clearance from microsomes, hepatocytes, and liver slices. Drug Metab. Rev. 29, 891922.[ISI][Medline]
Jordan, S. A., and Feeley, M. M. (1999). PCB congener patterns in rats consuming diets containing Great Lakes salmon: Analysis of fish, diets, and adipose tissue. Environ. Res. 80, S207S212.[ISI][Medline]
Kedderis, L. B., Mills, J. J., Andersen, M. E., and Birnbaum, L. S. (1993). A physiologically based pharmacokinetic model for 2,3,7,8- tetrabromodibenzo-p-dioxin (TBDD) in the rat: Tissue distribution and CYP1A induction. Toxicol. Appl. Pharmacol. 121, 8798.[ISI][Medline]
Kramer, H. J., van den Berg, M., De Lang, R. J., Brandsma, L., and Dejongh, J. (2000). Biotransformation rates of Ugilec 141 (tetrachlorobenzyltoluenes) in rat and trout microsomes. Chemosphere 40, 12831288.[ISI][Medline]
Krishnan, K., and Andersen, M. E. (1994). Physiologically based pharmacokinetic modeling in toxicology. In Principles and Methods of Toxicology (A. W. Hayes, Ed.), pp. 149188. Raven Press, Ltd., New York.
Lutz, R. J., Dedrick, R. L., Matthews, H. B., Eling, T. E., and Anderson, M. W. (1977). A preliminary pharmacokinetic model for several chlorinated biphenyls in the rat. Drug Metab. Dispos. 5, 386396.[Abstract]
Lutz, R. J., Dedrick, R. L., Tuey, D., Sipes, I. G., Anderson, M. W., and Matthews, H. B. (1984). Comparison of the pharmacokinetics of several polychlorinated biphenyls in mouse, rat, dog, and monkey by means of a physiological pharmacokinetic model. Drug Metab. Dispos. 12, 527535.[Abstract]
Matthews, H. B., and Dedrick, R. L. (1984). Pharmacokinetics of PCBs. Annu. Rev. Pharmacol. Toxicol. 24, 85103.[ISI][Medline]
Moir, D., Viau, A., Chu, I., Wehler, E. K., Morck, A., and Bergman, A. (1996). Tissue distribution, metabolism, and excretion of 2,4,4'-trichlorobiphenyl (CB-28) in the rat. Toxicol. Ind. Health 12, 105121.[ISI][Medline]
Murk, A. J., VandenBerg, J. H. J., Koeman, J. H., and Brouwer, A. (1991). The toxicity of tetrachlorobenzyltoluenes (Ugilec 141) and polychlorinated bifenyls (Arochlor 1254 and PCB 77) copared in Ah-responsive and Ah-nonresponsive mice. Environ. Pollut. 72, 5768.[ISI][Medline]
Obach, R. S. (1997). Nonspecific binding to microsomes: Impact on scale-up of in vitro intrinsic clearance to hepatic clearance as assessed through examination of warfarin, imipramine, and propranolol. Drug Metab. Dispos. 25, 13591369.
Ploeger, B. A., Meulenbelt, J., and DeJongh, J. (2000). Physiologically based pharmacokinetic modeling of glycyrrhizic acid, a compound subject to presystemic metabolism and enterohepatic cycling. Toxicol. Appl. Pharmacol. 162, 177188.[ISI][Medline]
Ryan, J. J., Levesque, D., Panopio, L. G., Sun, W. F., Masuda, Y., and Kuroki, H. (1993). Elimination of polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) from human blood in the Yusho and Yu-Cheng rice oil poisonings. Arch. Environ. Contam. Toxicol. 24, 504512.[ISI][Medline]
Tuey, D. B., and Matthews, H. B. (1980). Distribution and excretion of 2,2',4,4',5,5'-hexabromobiphenyl in rats and man: Pharmacokinetic model predictions. Toxicol. Appl. Pharmacol. 53, 420431.[ISI][Medline]
Van den Berg, M., De Jongh, J., Poiger, H., and Olson, J. R. (1994). The toxicokinetics and metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and their relevance for toxicity. Crit. Rev. Toxicol. 24, 174.[ISI][Medline]
van Haelst, A. G. (1996). Environmental chemistry of tetrachlorobenzyltoluenes. Thesis, University of Amsterdam, The Netherlands.
Wammes, J. I. J., Linders, S. H. M. A., Liem, A. K. D., and Gerrits, V. E. (1997). Ugilec 141, PCBs and Dioxins in Eel from the River Rur. RIVM, Bilthoven.
Wang, X., Santostefano, M. J., Evans, M. V., Richardson, V. M., Diliberto, J. J., and Birnbaum, L. S. (1997). Determination of parameters responsible for pharmacokinetic behavior of TCDD in female Sprague-Dawley rats. Toxicol. Appl. Pharmacol. 147, 151168.[ISI][Medline]
Yang, R., and Andersen, M. E. (1994). Pharmacokinetics. In Principles and Methods of Toxicology (A. W. Hayes, Ed.), pp. 4973. Raven Press, Ltd., New York.