Physiologically Based Pharmacokinetic Model for Developmental Exposures to TCDD in the Rat

Claude Emond*,{dagger}, Linda S. Birnbaum{dagger} and Michael J. DeVito{dagger},1

* National Research Council, Washington, District of Columbia 20001; and {dagger} National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received October 28, 2003; accepted February 15, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1: EQUATIONS FOR...
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent developmental toxicant in rodents, and these effects occur at exposures similar to background human body burdens. A physiologically based pharmacokinetic (PBPK) model can aid in quantitatively describing the relationship between exposure, dose, and response. The aim of this work was the development a PBPK model to describe the relationship between maternal TCDD exposure and fetal TCDD concentrations during critical windows of susceptibility in the rat. This PBPK model is a modification of an eight-compartment model that describes the adult female rat. The modified model reduces the compartments from eight to four maternal compartments (liver, fat, placenta and rest of the body). Activation of the placental compartment and a separate fetal compartment occurs during gestation. The systemic circulation connects the maternal compartments. The physiological and biochemical parameters were obtained from the literature. The model validation used experimental data from acute and subchronic exposures prior to and during gestation. The simulations predict the TCDD tissue concentrations of the maternal compartments within the standard deviation of the experimental data. The model overestimates the fetal concentrations by approximately a factor of two at low subchronic exposures, but does predict the fetal tissue concentrations within the range of the experimental data at the higher exposures. This model may provide a framework for the development of a human PBPK model to estimate fetal TCDD concentrations in human health risk assessments.

Key Words: PBPK; TCDD; rat; dioxin; developmental.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1: EQUATIONS FOR...
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent of the dioxin-like chemicals. These chemicals are found in all strata of the ecosystem (Birnbaum and DeVito, 1995Go; DeVito et al., 1995Go; Van Miller et al., 1976Go). TCDD and related chemicals induce numerous biochemical, physiological, and toxicological responses, including induction of CYP1A and 1B isoforms, modulation of growth factors and their receptors, immunotoxicity, carcinogenicity, and developmental toxicities (Birnbaum, 1995Go; Birnbaum and Tuomisto, 2000Go; DeVito and Birnbaum, 1994Go). The developmental toxicity of TCDD is well studied in rats, mice, and hamsters during the last 10 years (Gray et al., 1995Go, 1997aGo; Mably et al., 1992aGo,bGo,cGo; Theobald and Peterson, 1997Go). Initial studies by Mably et al. (1992aGo,bGo,cGo) reported that exposure to TCDD on gestation day 15 (GD15) results in a variety of reproductive alterations in male rats ranging from decreases in epididymal sperm to feminization of sexual behavior. Gray et al. (1997aGo,bGo) found permanent alterations in reproductive function in male rats and hamsters, including decreases in ejaculated sperm, following prenatal exposures to TCDD. Female rats exposed prenatally to TCDD have stunted development of the mammary glands and develop vaginal abnormalities that are described as a persistent vaginal thread (Fenton et al., 2002Go; Gray et al., 1997bGo; Hurst et al., 2002Go; Vorderstrasse et al., 2004Go). Similar effects occur in hamsters at slightly higher exposures (Wolf et al., 1999Go). Some of the developmental effects of TCDD in experimental animals are observed at or near background human exposures (DeVito et al., 2003Go). Markowski et al. (2001)Go found altered operant responding for motor reinforcement in rats at exposures similar to background human body burdens. A number of developmental reproductive effects also occur in mice, although at slightly higher TCDD exposures compared to rats (Theobald and Peterson, 1997Go).

Several studies in experimental animals examine the relationship between maternal TCDD exposure and fetal TCDD tissue concentrations. Li et al. (1995)Go found that fetal liver TCDD concentrations were similar to lean maternal tissue TCDD concentrations in rats receiving 5.6 µg TCDD/kg on GD18. Hurst et al. (1998Go, 2000aGo,bGo) demonstrated a uniform distribution of TCDD in the fetuses following low-dose acute and subchronic exposures to rats and found that fetal TCDD concentrations are similar to maternal blood and muscle TCDD concentrations. Abbott et al. (1996)Go also observed similar results in mice.

Humans are exposed to mixtures of dioxin-like chemicals. Van den Berg et al. (1987)Go found a linear relationship between maternal retention of dioxin congeners in the livers of the dams and offspring following oral exposures to fly ash containing a mixture of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). However, this study did not examine the pharmacokinetics of TCDD alone, and thus the potential for pharmacokinetic interactions is uncertain. The pharmacokinetics of TCDD is dose dependent, and it is likely that the exposure to other dioxin-like chemicals will influence the distribution and elimination of TCDD. Chen et al. (2002)Go examined the disposition of a mixture of dioxin-like chemicals in pregnant female rats and reports pharmacokinetic interactions between TCDD and other dioxin-like chemicals. A better understanding of the pharmacokinetics of TCDD in pregnant animals would aid in predicting the pharmacokinetic interactions of mixtures of dioxin-like chemicals.

Because the developmental toxicities of dioxin-like chemicals occur at such low exposures in experimental animals, understanding the relationships between exposure, dose, and response is important for accurate estimates of potential adverse human health risks. Physiologically based pharmacokinetic (PBPK) models quantitatively describe the relationship between exposure and tissue dose. PBPK models are mathematical descriptions of the physiological and biochemical processes involved in the absorption, distribution, metabolism, and elimination (ADME) of a chemical. Often, an understanding of the toxicological processes induced by a chemical is important in mathematically describing the pharmacokinetics of a chemical (Andersen and Dennison, 2001Go). PBPK models provide a quantitative means of extrapolating across species by substituting species-specific physiological and biochemical parameters into the model.

Several PBPK models have been published which describe the pharmacokinetics of TCDD in adult animals from different species (humans, mice, fish, and rats) (Leung et al., 1988Go, 1990Go; Maruyama et al., 2002Go; Nichols et al., 1998Go; Wang et al., 1997Go). In addition, Carrier et al. (1995aGo,bGo) described a simplified empirical pharmacokinetic model for rodents and humans. An important improvement in PBPK models for TCDD was the inclusion of an inducible hepatic TCDD binding protein (Leung et al., 1988Go). This protein was described as a low-affinity, high-capacity binding protein that is inducible by TCDD through activation of the Ah receptor. Several studies indicate that this protein is CYP1A2 (Diliberto et al., 1997Go, 1999Go; Poland et al., 1989Go). With few exceptions (Lawrence and Gobas, 1997Go; Maruyama et al., 2003Go), all other PBPK models published since the Leung model include an inducible hepatic TCDD-binding protein. While there is a preliminary human PBPK model describing gestational exposures to TCDD (Gentry et al., 2003Go), the validation of this model is limited due to the uncertainties in our understanding of past human exposures as they relate to the available data on TCDD tissue concentrations.

Interspecies extrapolation of pharmacokinetic models during gestation requires an understanding of the species differences in physiological changes occurring in both maternal and fetal compartments. In contrast to mature animals, rapid and complex changes occur during development from the embryonic through the neonatal period (Hayes, 1994Go). Because of these changes, the susceptibility of the organism can vary dramatically during these different life stages. TCDD induces a wide spectrum of effects on the developing reproductive system. Some of these effects appear to require in utero exposures only (Bjerke and Peterson, 1994Go; Lin et al., 2002Go), while the window of sensitivity for other responses remains uncertain. Studies examining windows of sensitivity for the developmental effects of TCDD have focused on determining whether the effects are due to lactational or in utero exposures. Hurst et al. (2000a)Go demonstrate that fetal TCDD concentrations on GD16 and GD21 provide good predictors of the developmental reproductive toxicity of TCDD (Hurst et al., 2000aGo). Determination of target tissue TCDD concentrations during these different developmental phases can aid in estimating potential adverse health effects from exposure to dioxins. PBPK modeling is a tool that should be used to predict the distribution of TCDD at different periods during development. The aim of this work was the development of a PBPK model for pregnant rats that predicts TCDD concentration in both maternal and fetal compartments throughout gestation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1: EQUATIONS FOR...
 REFERENCES
 
Model development. The model described in this paper is based on a PBPK model for TCDD in rats (Wang et al., 1997Go), which consists of eight compartments including blood, lung, kidney, skin, fat, liver, spleen and rest of the body. Lung, blood, and spleen are described as flow-limited compartments. Kidney, fat, and the rest of the body are described as diffusion limited. Wang et al. (1997)Go described liver and skin compartments as representing a more general case in which both flow and diffusion parameters influence tissue concentration, called a membrane-influenced condition. Validation of this model included several data sets from mice and rats.

Extrapolating a model with this many compartments to humans is problematic, due to the limited human data available. While some adult blood, breast milk, umbilical cord blood, and adipose tissue concentrations are available from the published literature, these tissues are rarely collected as maternal/infant pairs. Data from other tissues are not readily available. Thus, validating the model predictions in the different compartments in humans would be unlikely. The Wang model (1997)Go included lung, kidney, spleen, and skin compartments in order to predict enzyme induction in these tissues. In a model examining the distribution of TCDD during gestation, these compartments are not target tissues, nor do they significantly influence the pharmacokinetics of TCDD. Consequently, the PBPK model was simplified in order to more accurately represent the biological systems under study and describe the available human data.

Model representation. The model adapted for this analysis consists of four compartments (liver, fat, placenta, and rest of the body) for the dam and one compartment for the fetuses. Tissues in this model include those that have important roles in the pharmacokinetics and developmental toxicity of TCDD. Liver and fat were included in the model because they are involved in the metabolism and storage of TCDD and account for almost 80% of the body burden of TCDD (Carrier, 1991Go). A blood compartment was kept to describe the systemic circulation and because this tissue is readily sampled in humans. The rest of the body compartment was included in order to achieve mass balance. Each maternal compartment was interconnected by the systemic circulation (Fig. 1). Liver, fat, rest of the body (maternal), and placenta were described as diffusion limited. The kidney was included in the rest of the body compartment, and urinary clearance was adapted and compared to the Wang model (1997)Go. The Appendix includes all the equations used in this model.



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FIG. 1. Conceptual representation of PBPK model for rat developmental exposure to TCDD.

 
Hurst et al. (1998Go, 2000aGo,bGo) examined the fetal distribution of TCDD in rats following acute and subchronic exposures. In these studies, TCDD concentrations were similar between the fetal liver, urogenital tract, head, and rest of the body. Presently, our understanding of the molecular mechanisms of the developmental reproductive effects of TCDD is limited to the hypothesis that the binding and activation of the Ah receptor initiates a cascade of events leading to the effects. The model describes the distribution of TCDD to the fetuses using a single compartment. A more complex description of the fetal compartment would be of value in developing a pharmacodynamic model. However, there are no clear mechanisms to model at this time beyond binding of TCDD to the Ah receptor.

This model does not describe a blood flow to the fetal compartment. Rather, it assumes a simple diffusion of TCDD between the placental and fetal compartments. This diffusion was described using equivalent clearance rates from the placenta to the fetuses and a clearance rate from the fetuses to the placenta. The amount of TCDD that diffuses from the placenta to the fetuses maintains pseudo-equilibrium between the fetal lipid fraction and the rest of the fetuses. This equilibrium was described using a partition coefficient to estimate the concentration of TCDD leaving the fetal compartment, according to the equation shown in the Appendix. This partition coefficient is defined as the ratio of the TCDD concentration in the lipid fraction of the fetuses and the rest of the fetuses. During gestation, both the fetal and placental compartments are growing, although not at the same rate. Initially, placental parameters, such as tissue permeability, partition coefficient (tissue blood in placenta), and the clearance from placenta to fetuses or from fetuses to placenta, were visually fit to the experimental data of Hurst et al. (2000b)Go at a single dose of 0.2 µg TCDD/kg on GD15. A formal optimization using the ACSL OptimizeTM module of ACSL MathTM followed the visual fitting. ACSL OptimizeTM uses a maximization of the log likelihood function in the optimization process.

A pseudo-compartment, which does not provide a physiological representation of the process, describes the oral absorption. The absorption of TCDD is described as occurring through both the portal and the lymphatic circulation (Fig. 1). The lymphatic circulation was described by Wang et al. (1997)Go, based on the work of Roth et al. (1993)Go. The present description of this simplified PBPK model represents the most important compartments for an adequate description of the pharmacokinetics of TCDD. Comparison of this reduced model with the full Wang model demonstrated that the reduced model prediction varies no more than 10 to 15%, depending on the tissue, time. and dose (data not shown).

A number of pharmacokinetic studies have demonstrated a dose-dependent sequestration of TCDD in the liver (Abraham et al., 1988Go; DeVito et al., 1998Go; Diliberto et al., 1997Go, 1999Go; Poland et al., 1989Go). Several studies indicate that CYP1A2 is the inducible binding protein (Diliberto et al., 1997Go, 1999Go; Poland et al., 1989Go). TCDD induces CYP1A2 through an Ah receptor-mediated mechanism. The CYP1A2 induction and binding of TCDD is described by the model in the maternal hepatic compartment. These processes were not described in the fetal compartment for several reasons. Data from Hurst et al. (2000aGo,bGo) demonstrates a lack of hepatic sequestration in fetal livers and that the concentration in the fetus is uniform. Comparisons of the maternal and fetal TCDD concentrations from the Hurst studies demonstrate that the maternal liver has TCDD concentrations 100- to 1000-fold higher than the fetal liver. These data are consistent with the lack of inducible expression of CYP1A2 in the fetal liver (Borlakoglu et al., 1993Go). Maternal alterations in CYP1A2 during pregnancy appear minor (Borlakoglu et al., 1993Go). In humans, expression of hepatic CYP1A2 does not appear until approximately 3–4 months of age (Oesterheld, 1998Go; Sonnier and Cresteil, 1998Go).

Model parameterization. All parameters for nonpregnant animal were adapted from Wang et al. (1997)Go. For the nonpregnant animal, only the fat, liver, and rest of the body compartments were activated. In these simulations, the tissue volume and blood flow rates vary in proportion to body weight. Parameters for growth of the placental, blood and fetal compartments as well as alterations in blood flow rates are based on the data of O'Flaherty (1994)Go and Buelke-Sam et al. (1982aGo,bGo) and were incorporated into the PBPK model at the beginning of gestation. The placenta grows from zero to 0.55 g for each fetus (Fig. 2a). The mean number of fetuses per litter was 10. Placental distribution of TCDD was described as diffusion limited. Placental blood flow was assumed to increase from zero to 10% of cardiac output according to the relationship described in Figure 2b. Adipose tissue volume increases during gestation were described previously (Fig. 2c) (Buelke-Sam et al., 1982aGo,bGo; Fisher et al., 1989Go; Gentry et al., 2002Go; Mattison et al., 1991Go; O'Flaherty, 1994Go; O'Flaherty et al., 1992Go; Sikov and Thomas, 1970Go). Fetal growth was also incorporated into the model, assuming that each fetus weighed 6 g on GD22, with a total fetal compartment of 60 g and assuming 10 fetuses/litter (Fig. 2d). Exponential equations were used for these growing compartments except for the maternal adipose tissue compartment, where published literature suggests a linear increment (see expression in legend of Fig, 2) (Fisher et al., 1989Go; O'Flaherty, 1994Go; O'Flaherty et al., 1992Go). The remaining tissues still grow in proportion to the body weight. The rate of growth of the rest of the body compartment is adjusted in proportion to the growth rate of the placental and fat compartments during gestation in order to maintain mass balance.



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FIG. 2. Growth rates for physiological changes occurring during gestation. (a) Placental growth during gestation (calculated for 10 placenta). Experimental data came from Sikov (1970)Go. Mathematical expression used:

Physiological changes during pregnancy obtained from the literature. (b) Blood flow rate in placental compartment during gestation. Experimental data came from Buelke-Sam et al. (1982aGo,bGo). Mathematical expression used:

(c) Fat fraction of body weight during gestation. Experimental data came from Fisher et al. (1989)Go. Mathematical expression used:

(d) Fetal growth during gestation (data and model represent 10 fetuses). Experimental data from Sikov (1970)Go. Mathematical expression used:

 
Model simulation and validation. The PBPK model was developed with algebraic and differential equations describing the kinetics of TCDD using the commercially available software ACSLTM (Advanced Continuous Simulation Language, Aegis Corporation. Huntsville, AL). The optimization of fetal and placental clearance and fetal partition coefficient were optimized using ACSL Optimize, a module of ACSL MathTM. Five experimental studies were used to validate this model and are described below. A more detailed model description is available in the Appendix, and the physiological parameters used in the model are in Table 1.


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TABLE 1 Physiological Parameters Used in the PBPK Models for Nonpregnant and Pregnant Rats

 
Experimental Data Used for Validation
Acute exposure in nonpregnant rats. Female Sprague-Dawley rats (age, 8 weeks; 250 g) received a single dose of 10 µg 3H-TCDD/kg of body weight (Santostefano et al., 1996Go). Tissue samples were collected at 0.5, 1, 3, 8, and 24 h, 7 days, 14 days, and 35 days after exposure. Three to five animals/group were used.

Subchronic in nonpregnant rats. Male Wistar rats were treated using a loading dose/maintenance regimen with 14C-2,3,7,8-TCDD (Krowke et al., 1989Go). Rats were exposed to 25 ng TCDD/kg body weight followed by weekly maintenance doses of 5 ng TCDD/kg body weight by gavage. Liver, fat, thymus and kidney samples were collected at 1, 2, 3, 4, 6, 8, 10, 12, 14, and 22 weeks after the initial exposure and analyzed for TCDD concentrations. Only two rats were used per time point, and the data are presented separately for each animal.

Acute exposure during gestation in rats. In the rat, the prenatal period appears to be the window of sensitivity for many of the developmental reproductive effects of TCDD (Gray et al., 1997aGo,bGo). Only a few studies have determined maternal and fetal TCDD concentrations. There are several studies examining TCDD concentrations on postnatal day 21 or later, following gestational exposures (Faqi et al., 1998Go; Nohara et al., 2000Go; Ohsako et al., 2001Go). Li et al. (1995)Go determined fetal rat TCDD concentrations on GD19 and 20, following an exposure to 5.6 µg TCDD/kg on GD18. Van den Berg et al. (1987)Go and Chen et al. (2002)Go determined fetal concentrations of TCDD after exposing pregnant rats to a mixture of dioxin-like chemicals. The studies using the mixtures are not used in this modeling exercise because the coexposure to other chemicals altered the pharmacokinetics of TCDD (Chen et al., 2002Go). Due to the limited data on fetal TCDD concentrations available, the data of Hurst et al. (1998Go, 2000aGo,bGo) and Li et al. (1995)Go were used to develop and validate the model.

Hurst et al. (2000b)Go exposed pregnant Long-Evans rats (200–250 g) to a single dose of 0.05, 0.2, 0.8, or 1.0 µg 3H-TCDD/kg on GD15. Dams were terminated on GD16 and GD21, and liver, fat, blood, kidney, skin, and placenta were collected from the dams. The fetuses were dissected, and liver, urogenital tract, head, and remainder of the carcass were analyzed for TCDD concentrations. Five animals per group were used. In a preliminary study, Hurst et al. (1998) exposed Long Evans rats to 1.15 µg 3H-TCDD/kg on GD8 or GD15 and collected maternal and fetal tissues on GD9, GD16, and GD21. Due to the limited experimental data available, the data from the 0.2 µg TCDD/kg dose was used to optimize the model, and the remaining data from Hurst et al. (1998Go; 2000bGo) and the data from Li et al. (1995)Go were used to validate the model.

Subchronic exposure during gestation in rat. In a subchronic study, Hurst et al. (2000a)Go exposed female Long-Evans rats (200–300 g) to either, 1, 10, or 30 ng of 3H-TCDD/kg/d for 5 days/week for 13 weeks by gavage. After 13 weeks, females were mated, and GD0 corresponded to the date of a positive sperm plug. Exposures were continued daily from GD0 through parturition. Liver, fat, blood, kidney, skin, and placenta were collected from the dams on GD9, GD16, and GD21. The fetuses were dissected, and liver, urogenital tract, head, and remainder of the carcass were analyzed for TCDD concentrations.

Model evaluation and sensitivity analysis. This model was evaluated using the discrepancy index (Krishnan et al., 1995Go). This index measures the difference between PBPK model simulations and experimental data. This test can also compare model simulations. The lower the discrepancy index, the higher the confidence in the model. The discrepancy index was calculated to evaluate the ability of the reduced model to simulate hepatic and adipose tissue concentrations in rats exposed to single oral dose of 10 µg TCDD/kg (Santostefano et al., 1996Go). In addition, the discrepancy index was used to determine the differences between the Wang et al. model (1997)Go and the reduced model. The discrepancy index was also calculated to evaluate the model predictions during gestation by lumping either tissues or exposures levels. For example, a discrepancy index was calculated for an individual compartment at all dose levels and time points. A second discrepancy index was calculated by lumping all time points and tissues for a specific exposure. Discrepancy indices were calculated separately for the acute and subchronic exposures. While the discrepancy index itself is uninformative, it does allow for objective comparisons of fits across models, exposure scenarios and tissues.

During model simulations, it is important to establish the sensitivity of the parameters to small changes. Each parameter of this model was tested for sensitivity. This evaluation consisted of varying each parameter by a factor of ± 10%. Sensitivity analysis was performed at an exposure of 1 µg TCDD/kg at GD15, and TCDD concentrations in the fetal compartment were modeled at the end of gestation corresponding to GD21. Because distribution is dose dependent, a low exposure of 0.05 µg of TCDD/kg was also simulated. A comparison between acute and subchronic exposures was done to understand the influence of repeated exposures on estimates of maternal blood and fetal concentrations at an exposure of 1 ng TCDD/kg/day by gavage. These different comparisons provide information about the behavior of the model under different exposure conditions.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1: EQUATIONS FOR...
 REFERENCES
 
Comparison of the Reduced PBPK Model to the Wang Model
Initially, the Wang et al. model (1997)Go was reduced to the model represented in Figure 1 without the placental and fetal compartments. The reduced model was visually optimized for the TCDD dissociation constant for CYP1A2 (KDLI2) using data from a single oral exposure of 10 µg TCDD/kg in female rats (Santostefano et al., 1996Go). Based on this optimization, KDLI2 was increased to 0.04 nmol/ml from 0.03 nmol/ml. All other parameters in this model were identical to those in the Wang et al. model (1997)Go.

The outputs of the Wang model (Wang et al., 1997Go), and the reduced model for the distribution of TCDD in rats were similar, with the exception of a couple of minor differences. The maximal liver concentration occurring in the absorption phase was lower in the Wang model as compared to the reduced model, from 4.4 to 4.8% dose/g, respectively (data not shown). This is due, in part, to slight differences in the absorption expressions used by Wang et al. (1997)Go compared to the present model, although grouping of the organs into fewer compartments also played a minor role. After the absorption phase, distribution and elimination were similar between the two models. In addition, in the reduced model, the TCDD affinity constant for CYP1A2 binding was increased from 0.03 nmol/ml to 0.04 nmol/ml. This first value was obtained by Wang et al. (1997)Go after visual optimization of their parameters. By changing this value, a slightly better fit to the experimental data for liver and fat was obtained compared to the fit found by Wang et al. (1997)Go.

The discrepancy index was calculated for model simulations of the hepatic and adipose tissue concentrations in rats exposed to a single oral dose of 10 µg TCDD/kg (Santostefano et al., 1996Go) (Table 2a). Using an affinity constant for CYP1A2 of 0.03 nmol/ml, the reduced model gave discrepancy indices of 0.22 and 0.40 for liver and adipose, respectively. When this parameter is increased to 0.04 nmol/ml, the discrepancy indices were reduced to 0.06 and 0.34 for liver and adipose. A comparison between the Wang model and the reduced model gave a discrepancy index of 0.19 for liver and 0.15 for adipose.


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TABLE 2 Discrepancy Index Analysis

 
Simulation of Acute TCDD Exposure in Nonpregnant Rats
This simulation reproduced an acute exposure of a single oral dose of 10 µg TCDD/kg of body weight in rats as described by Santostefano et al. (1996)Go. Time-course samples were taken starting at 30 min and lasting until 850 h after the initial exposure. Simulations were conducted for 900 h. Figure 3 compares the simulations obtained with the reduced model to the experimental data (Santostefano et al., 1996Go). The model predictions are within the standard deviation of the experimental data except for the last time point for blood concentrations, where the model underestimates the concentration by a factor of approximately 3. Peak TCDD concentrations in the liver compartment are higher than in the fat compartment. This is consistent with the binding of TCDD to hepatic CYP1A2 and its sequestration in the liver (Diliberto et al., 1997Go, 1999Go). The model predicts that the initial ratios of the TCDD concentrations in liver to fat are higher than at the later time points, consistent with the experimental data (Fig. 3).



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FIG. 3. Experimental data and the model predictions of TCDD tissue concentrations in rats following an acute exposure to TCDD. TCDD tissue concentrations (% administered dose/g tissue) were measured or estimated in blood, fat, liver and rest of the body of rats after a single dose of 10 µg TCDD/kg by gavage. Symbols represent experimental data for blood (open circle), fat (dark square), liver (dark triangle) and muscle (open square) (Santostefano et al., 1996Go). The experimental data for muscle concentrations was used as a surrogate for the rest of the body compartment in the reduced model. Each point represents mean ± SD for 4 to 5 rats per group per time point. Lines represent model simulations.

 
Simulation of Subchronic TCDD Exposure in Nonpregnant Animals
The data of Krowke et al. (1989)Go was used by Wang et al. (2000)Go to examine the ability of the model to predict subchronic exposure regimens. Male Wistar rats were exposed to TCDD by subcutaneous injection in a loading dose/maintenance dose regimen (Krowke et al., 1989Go). A loading dose of 25 µg TCDD/kg was followed with a weekly maintenance dose of 5 µg TCDD/kg for 22 weeks. Concentrations of TCDD (ng/g of tissue) were reported for several tissues. Simulations with the reduced PBPK model were performed over a 24-week period and compared to experimental data (Figure 4). The simulations of the reduced model are consistent with the results of the data from Krowke et al. (1989)Go. The model predicts a rapid approach to pseudo-steady-state concentrations during the first few weeks after the initial loading dose. While there are slight differences between the model and the data, it should be noted that the data consists of only two animals/time point and is quite variable. The current fits are consistent with those obtained by the Wang et al. (2000)Go PBPK model, given the variability in the Krowke et al. (1989)Go data set.



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FIG. 4. Distribution of 14C-TCDD in rats following a loading dose/maintenance dose exposure scenario. Rats were exposed to an initial dose of 25 µg of TCDD/kg followed by a weekly maintenance dose of 5 µg/kg for 22 weeks (Krowke et al.,1989Go). Lines represents concentrations simulated with the PBPK model for liver and fat. Symbols represent experimental data for liver (triangle) and fat (square) and each symbol represents data from an individual animal.

 
Development of the Pregnant Rat Model
Following the initial validation of the reduced model in nonpregnant rats, placental and fetal compartments were added to the model. The tissue permeability, the placental tissue:blood partition coefficient, and the placental to fetal clearance were optimized using ACSL Optimize (ACSL Math, version 2.1). This uses a maximization of the log likelihood function as described by Steiner et al., 1990Go). The experimental data used in this optimization came from Hurst et al. (2000b)Go, who exposed Long Evans rats to 0.05, 0.20, 0.80, or 1.0 µg TCDD/kg on GD15 by oral gavage. On GD16 and 21, animals were killed, and maternal liver, fat, blood, placenta and fetal liver, urogenital tract, head, and rest of the body were dissected and analyzed for TCDD concentration. The 0.20 µg TCDD/kg dose was used in the formal optimization.

In Figures 5a (blood), 5b (fat), 5c (liver), 5d (placenta), and 5e (fetuses) the experimental data and model simulations for 0.05, 0.20, 0.80, and 1.0 µg TCDD/kg, are presented. At these exposures, induction of CYP1A2 and hepatic sequestration of TCDD are present in the maternal liver, as demonstrated by liver/fat TCDD concentration ratios greater than 1. However, at an exposure of 0.05 µg TCDD/kg, sequestration by CYP1A2 is less pronounced compared to the other exposure levels. The model slightly underpredicts the TCDD concentrations in all tissues by a factor of 2 or less at the 0.05 µg TCDD/kg dose at GD21. Blood concentrations are underpredicted by up to a factor of 3 at all concentrations and time points. Liver concentrations are predicted within 20% of the experimental data, with the exception of the 0.05 µg TCDD/kg dose on GD21. Placental and fetal concentrations are predicted within the standard deviation of the experimental data except at the 0.05 µg TCDD/kg dose on GD21. Model predictions for adipose tissue are within the standard deviation of the experimental data except for the 0.2 and 0.05 µg/kg exposures on GD16 and 21. These predictions are within a factor of 2 compared to the experimental data.





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FIG. 5. Time course of TCDD distribution in (a) blood, (b) adipose tissue, (c) liver, (d) placental and (e) fetal compartments after a single oral exposure to 0.05, 0.2, 0.8, or 1.0 µg TCDD/kg at GD15 in rats. These simulations were compared to experimental data (Hurst et al., 2000bGo). Symbols represent the mean ± standard deviation of the experimental data from animals exposed to 0.05 (dark square), 0.2 (dark triangle), 0.8 (dark circle), or 1.0 µg TCDD/kg (lozenge). Tissue concentrations are expressed in pg TCDD/g tissue. Lines represent model simulations.

 
In an initial study, Hurst et al. (1998) administered a single oral exposure of 1.15 µg TCDD/kg to rats on GD8 and determined tissue concentrations on GD9, GD16, and GD21. Simulations of this exposure result in predictions of fetal concentrations in GD21 and maternal tissue concentrations, including the placental concentrations, within the range of the experimental data (data not shown). However, the model overestimates the fetal concentrations on GD9 and GD16 by a factor of almost 10.

Evaluation of the model continued by simulating the experimental data of Li et al. (1995)Go, who exposed pregnant Sprague-Dawley rats on GD18 to a single iv dose of 5.6 µg TCDD/kg. TCDD concentrations were determined on GD19 and 20 in maternal blood, liver, and fat and fetal liver. The model overestimated maternal liver TCDD concentrations by a factor of approximately 2 (Table 3). Maternal fat TCDD concentrations are predicted within 10–30% of the experimental value. TCDD blood concentrations estimated by the model were approximately 3 times lower than the serum TCDD concentrations determined experimentally. The difference in the model predictions of blood concentrations and experimentally determined serum concentrations may be explained by the distribution of TCDD between whole blood and serum (Patterson et al., 1987Go). Li et al. (1995)Go determined fetal liver concentrations. Based on the data of Hurst et al. (2000aGo,bGo) our model assumes that the fetus is a well-stirred compartment and that fetal liver concentrations should be equivalent to our model estimates of the concentrations in the fetal compartment. Our model predicts fetal concentrations approximately two-fold lower than the experimental data of Li et al. (1995)Go (Table 3).


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TABLE 3 A Comparison Between Li et al. (1995)Go Tissue Concentration Data and the Model Simulation

 
Simulation of Subchronic TCDD Exposure Prior to Mating and Continuing Throughout Gestation
Female Long Evans rats, were exposed by gavage to 1, 10, or 30 ng of 2,3,7,8-TCDD/kg, 5 days/week for 13 weeks prior to mating (Figs. 6a, through 6e) (Hurst et al., 2000aGo). Daily exposures were initiated after mating and continued through parturition. The model simulations and the data indicate that the exposure paradigm results in near steady-state tissue concentrations in the maternal tissue compartments throughout gestation. In the maternal tissue compartments, the model is better at predicting tissue concentrations at the higher exposures. The model tends to underestimate the tissue concentrations for the lower exposures by up to factors of 3 to 4 (Figs. 6a through 6e).




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FIG. 6. Time course of TCDD distribution in (a) blood, (b) adipose tissue, (c) liver, (d) placental, and (e) fetal compartments. These simulations were compared to experimental data (Hurst et al. 2000aGo). Female rat were exposed to 1, 10, and 30 ng/kg, 5 days/week 13 weeks prior to mating followed by daily exposures through parturition. Concentrations of TCDD were determined on GD9, GD16 and GD21. Symbols represent 1 (square), 10 (triangle), and 30 (circle) ng TCDD/kg/d. Experimental data correspond to the mean concentration ± SD. Concentrations were expressed in pg of TCDD/g of tissue. Lines represent model simulations.

 
At the early time points, embryo and placental concentrations are negligible based on the model predictions. Embryo and placental concentrations rise rapidly between GD5 and GD6. Placental concentrations are at steady state starting at approximately GD7, which is consistent with the experimental data on GD16 and GD21. The model predicts that the embryo/fetal tissue concentrations are not at steady state and vary by approximately a factor of 2 between GD9 through GD21 (Fig. 6e). Fetal concentrations are predicted to be lower than placental concentrations in the subchronic exposure. At the end of gestation, the model predicts that fetal concentrations are slightly lower than placental concentrations. The experimental data on GD16 and GD21 indicated that fetal and placental concentrations are equivalent, and the PBPK model approximates these concentrations.

Discrepancy indices were calculated for either tissues or dose level in the acute and subchronic exposure data from Hurst et al. (2000aGo,bGo). Discrepancy indices ranged from 0.08 for placenta to 0.54 for maternal blood in the acute exposures (Table 2b). The discrepancy indices ranged from 0.18 for liver to 0.57 for the fetal compartment in the subchronic exposures. Discrepancy indices for the maternal compartments in the acute and subchronic simulations are similar. The discrepancy indices were lower by factors of two and four in the acute exposures for fetal and placental concentrations, respectively. When the discrepancy indices were calculated for the different dose levels, the acute exposure had similar discrepancy indices at all dose levels except for the 0.8 µg TCDD/kg dose (Table 2c). In the subchronic exposure, the discrepancy index was dose dependent indicating that the model simulates the higher exposures better than the lower exposures (Table 2c).

Sensitivity Analysis
Sensitivity analysis was performed on all parameters in this PBPK model for both acute and subchronic exposures. However, to simplify the presentation of the analysis only parameters that resulted in a normalized sensitivity coefficient of >+0.2 or <–0.2 are presented and discussed. In some cases, there was a dose dependency in the normalized sensitivity coefficients. In these cases, we report the normalized sensitivity coefficient for all sensitivity analyses.

The acute exposure scenario was analyzed at two dose levels, 0.05 and 1.0 µg TCDD/kg, at GD15, and the sensitivity analysis was done on fetal tissue concentrations on GD21 (Table 4). For the acute exposure scenarios, dose-dependent effects on the sensitivity analysis were observed (Table 4). At the low dose, absorption constant (KABS), gastric transit time constant (KST), elimination constant (KBILE_LI), AhR binding concentration in placenta (PLABMAX), partition coefficient fat:blood (PF), partition coefficient placenta:blood (PPLA), and liver volume fraction of body weight (WLI0), are the most sensitive parameters (Table 4). As expected, variations in parameters describing the induction of CYP1A2 had little impact on estimated fetal TCDD concentrations at the low dose exposure. The variations in other parameters resulted in sensitivity coefficients of less than ±0.2. Similar results were observed at the higher exposure (Table 4). However, at the high dose, 1 µg TCDD/kg body weight, the degradation constant of CYP1A2 had a normalized sensitivity coefficient of 0.2 (Table 4). Changing the degradation constant of CYP1A2 by 10%, results in a 2% difference in the fetal TCDD concentrations (Table 4).


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TABLE 4 Sensitivity Analysis of the PBPK Model Parameters

 
In the subchronic exposure scenario, the analysis was performed at an exposure of 0.001 µg TCDD/kg/day, 5 days/week for 13 weeks followed by daily exposure during mating and gestation (Table 4). Fetal tissue concentrations on GD21 were used as reference for this sensitivity analysis. The sensitivity analysis for the subchronic exposure was similar to the results for the low dose acute exposures (Table 4), with the exception that the fat partition coefficient (PF) was less sensitive in the subchronic exposure than in the acute exposure (Table 4).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1: EQUATIONS FOR...
 REFERENCES
 
A number of PBPK models describe the absorption, distribution, elimination, and metabolism of TCDD in adult rats and mice. The more recent models incorporate an Ah-receptor-mediated induction of a hepatic TCDD binding protein (Andersen et al., 1993Go; Kohn et al., 2001Go; Wang et al., 1997Go). A more detailed description comparing these models is presented in Wang et al. (1997Go, 2000Go). The present study modified the Wang et al. (1997)Go PBPK model to describe the distribution of TCDD between the dam and fetal rat. This simplified PBPK model can be used to simulate TCDD tissue concentrations in both nonpregnant adult female rats and pregnant rats. This model could also be used for acute and subchronic exposure scenarios and can serve as the basis for the development of a PBPK model to describe TCDD distribution in humans.

Because gestation is a dynamic process, several parameters are also dynamic. For example, changes in fetal and placental growth, maternal adipose tissue mass fraction, and placental blood flow during gestation are included in the model. Incorporation of these dynamic processes is important in order to predict TCDD concentrations throughout pregnancy. The changes in maternal parameters, such as adipose mass fraction, are not as dramatic as the changes in fetal parameters. Thus, in the subchronic exposures, maternal tissue concentrations do not significantly vary during pregnancy. In contrast, because of the dynamic process of development the model predicts large variations in embryo/fetal TCDD concentrations from GD6-10, even when maternal concentrations are in pseudo-steady-state. This dynamic process also increases the uncertainty in parameter estimates such as the partition coefficient in the embryos compared to the late stage fetuses and the permeability between placenta and fetuses. For example, on GD9, the embryo is physiologically different from the fetus on GD19, and one would expect this difference to influence the partition coefficient. It is possible that these parameters will change during gestation.

The present model assumes that placental and fetal partition coefficients are constant during gestation and exposures. Chen et al. (2002)Go report that on GD16 and 21, the total lipid content of the placental and fetal compartments is constant. In the present model, the greatest changes in fetal TCDD concentrations occur during the early embryonic period. This is also the period of development where there is limited data available on TCDD concentrations or fetal and placental lipid composition. Thus, model estimates of TCDD tissue concentrations earlier than GD16 must be viewed cautiously.

Model Reduction
The required complexity of a PBPK model depends on the question that the model is attempting to address. Our overall goal was to develop a PBPK model that estimates human fetal TCDD concentrations under a variety of exposure scenarios. Because of the limited human data available to validate such a model, one approach would be to develop a simplified human PBPK model based on a validated rodent PBPK model. The present model is a reduction of the rat PBPK model developed by Wang et al. (1997)Go and incorporates only tissue compartments that significantly influence the distribution of TCDD. While the model is reduced, it provides similar fits to the data as that of the full model. This observation is due to the fact that the liver and fat compartments represent around 80 to 95% of the TCDD body burden (Carrier, 1991Go; DeVito et al., 1998Go; Diliberto et al., 2001Go), and these two compartments are represented in both models. Comparisons of the reduced model with that of Wang et al. (1997)Go demonstrate no significant difference between the TCDD tissue concentration profile curves (Table 2) for nonpregnant female rats for both acute and subchronic exposures. In addition to reducing the number of compartments in the earlier model, the affinity constant of TCDD for CYP1A2 was changed from 0.03 to 0.04 (nmol/ml). This change results in slightly better fits compared to the experimental data (Fig. 3) as demonstrated by the discrepancy index (Table 2).

Maruyama et al. (2003)Go take a different approach to model reduction in their pharmacokinetic model for TCDD and related chemicals for use in assessing the potential adverse reproductive effects of these chemicals. They developed a human PBPK model for TCDD and dioxin-like chemicals that describes seven maternal compartments including blood, liver, fat, skin, kidney, richly perfused, and muscle. In their assessment of the reproductive risk associated with TCDD exposure, these authors used the richly perfused compartment as a surrogate for fetal tissue. This model does not incorporate Ah-receptor-mediated hepatic sequestration of TCDD or the physiological changes occurring during pregnancy.

Validation of the Gestational Rodent Model
The reduced model incorporates placental and fetal compartments. A PBPK model was developed, and parameter optimization was performed using a single dose level (0.2 µg TCDD/kg on GD15) from the experimental data of Hurst et al. (2000b)Go. The model was validated using the experimental data from rats exposed to 0.05, 0.8, and 1.0 µg TCDD/kg on GD15 (Hurst et al., 2000bGo). The model provided adequate fits to this data (Figs. 5a through 5e). In contrast to adult rats, fetal distribution of TCDD appears uniform. Based on this experimental data, a single fetal compartment seems to be adequate to describe fetal TCDD concentrations. This is due to the lack of CYP1A2 induction in fetal liver. The fetal compartment may be viewed as a nonsequestering organ (Hurst et al., 2000bGo). It should be noted that parameters describing the binding and induction of CYP1A2 and Ah receptor binding were not altered between the pregnant and nonpregnant models. This suggests that these parameters were not altered by pregnancy.

Simulation of fetal TCDD concentrations following subchronic exposures provided good fits to the data of Hurst et al. (2000a)Go. Similar tissue concentration profiles were observed in the simulations of the subchronic exposure compared to those presented for acute exposures. In the acute exposures, the model predicts lower fetal TCDD concentrations compared to placental TCDD concentrations from GD15 to parturition. The subchronic model also predicts lower fetal TCDD concentrations compared to placental TCDD concentrations until approximately GD13. After that, the TCDD concentration in the fetuses was similar to placental TCDD concentration. Fetal TCDD concentrations decrease during the latter gestation period in both the acute and subchronic exposure scenarios as predicted by the model.

There are differences in the shape in the tissue concentration versus time curves between the fetal and placental compartment. In early time points, the placental and fetal curves are probably driven by the absorption description as well as by the placental equilibrium with blood. The differences in the concentration versus time profiles for these tissues are most likely due to the differences in the rate of fetal and placental growth. Fetal growth rates are significantly greater than the placental growth rate (Figs. 2a and 2d). Changing the fetal growth rate to a linear model results in similar tissue concentration versus time profiles for the fetal and placental compartments (data not shown). These results demonstrate the importance of understanding the different growth rates of these dynamic processes and their impact on estimating tissue concentrations.

The model predicts fetal concentrations within a factor of two at all exposure scenarios and time points except for the Hurst et al. (1998)Go data from the GD8 exposures. The model overestimates the tissue concentrations on GD9 and GD16 by almost an order of magnitude. The model does predict the fetal concentrations within the range of the experimental data in the subchronic exposures and acute the acute exposures on GD15. These results indicate that the description of the early periods of placental, embryo, and fetal development may need improvement.

Sensitivity Analysis
Sensitivity analysis is a useful approach for understanding how the different parameters influence the model predictions. In addition, sensitivity analysis allows for a quantitative comparison of the influence of the different physiological processes described by the model. The sensitivity of the model to parameter variation is qualitatively similar for both acute and subchronic exposures. The most sensitive parameters describe chemical-specific phenomena. For example, at higher acute TCDD exposures, parameters describing maternal CYP1A2 induction and binding of TCDD to maternal CYP1A2 have a greater influence on fetal TCDD concentrations than the at the lower exposures. This is consistent with the dose-dependent induction of CYP1A2 by TCDD. CYP1A2 binds and sequesters TCDD and related chemicals in hepatic tissue (Diliberto et al., 1997Go, 1999Go) At higher levels of CYP1A2 induction, the maternal liver would sequester a greater percentage of the TCDD dose, decreasing TCDD distribution to extrahepatic tissue, including the fetal compartment. These results could be important when extrapolating this model to other species, including humans.

Clewell et al. (2003)Go and Sweeney et al. (2001)Go also perform sensitivity analyses for their developmental models. The most sensitive parameters in these studies describe chemical-specific processes. Thus, while these models describe gestational exposures, sensitivity analysis indicates that the models are most sensitive to small changes in chemical-specific parameters. Direct comparisons between these sensitivity analysis results should be viewed cautiously for several reasons. The molecular mechanisms of the developmental effects of perchlorate are better understood than those for TCDD. Clewell et al. (2003)Go developed a much more complex fetal model that incorporates fetal blood flow and several other compartments including the fetal thyroid gland, the target tissue for the effects of perchlorate. TCDD and perchlorate are also very different chemically. Sweeney et al. (2001)Go incorporate physiological changes during gestation in the maternal compartment but do not have specific placental or fetal compartments. The rapidly perfused compartment is a surrogate for fetal concentrations of 2-methoxyethanol and 2-ethoxyethanol in the Sweeney et al. model (2001)Go. Because of these differences, comparisons of sensitivity analysis of these models may not provide insight into a broader discussion on the use and application of PBPK models during development.

Conclusion and Significance for Human Risk Assessment
Pharmacokinetic factors are important in understanding toxicity processes, particularly during development. The duration of critical windows of sensitivity is an important determinant in developmental toxicity (Wilson, 1965Go). A number of reports demonstrate both male and female developmental reproductive toxicity of TCDD. Critical windows of exposure appear to be around GD15 (Gray et al., 1997bGo; Hurst et al., 2000bGo) in the rat for some of these effects such as decreases in epididymal and ejaculated sperm counts, vaginal opening, and the appearance of persistent vaginal threads. In the present study, the PBPK model suggests that the fetal TCDD concentration may vary with developmental stage. Understanding the relationship between the critical window of sensitivity and the influence of developmental stage on the distribution of TCDD is important in estimating the concentration/response relationship for the adverse effects of TCDD.

The PBPK model developed in this report describes the distribution of TCDD in pregnant rats. Limited data sets were available to develop and validate this model. Hurst et al. (2000aGo,bGo) have presented dose-response and time-course data for fetal concentrations of TCDD following acute and subchronic exposures. The limited data on fetal concentrations of TCDD in rats suggest that extrapolation of this model to humans should be done cautiously. Efforts should be made to develop other data sets in other strains and species in order to increase confidence in the use of this model prior to human extrapolations. In addition, earlier gestational time points will be useful in further validating this model.


    APPENDIX 1: EQUATIONS FOR THE RAT PBPK GESTATIONAL MODEL
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1: EQUATIONS FOR...
 REFERENCES
 
Variation of Body Weight with Age

All other tissue growth rates are presented in the legend for Figure 2.

Cardiac Output

A factor of 60 corresponds to the conversion of min to h, and 1000 is conversion of body weight from g to kg.

Blood Compartment

Tissue Compartment (Fat, Rest of the Body)

  1. Tissue-blood subcompartment


  2. Tissue cellular matrices


Liver Tissue Compartment (Liver)

  1. Tissue-blood subcompartment


  2. Tissue cellular matrices


Free TCDD Concentration in Liver

Concentration Bound to Ah Receptor in Tissue (Liver and Placenta)

All others induction processes and equations have been described and presented in Wang et al. (1997)Go.

Placenta Tissue Compartment

  1. Tissue-blood subcompartment


  2. Tissue cellular matrices


Free TCDD Concentration in Placenta

Dioxin Transfer from Placenta to Fetuses

Dioxin Transfer from Fetuses to Placenta

Fetal Dioxin Concentration (Fetuses = 10 Per Litter)

Gastro-Intestinal Absorption and Distribution of TCDD to the Portal Lymphatic Circulation. Amount of TCDD remaining in lumen cavity

Lumen: Amount of TCDD remaining in the GI tract (nmole)
Intake: Rate of intake of TCDD during a subchronic exposure (nmol/hour)

Amount of TCDD Eliminated in the Feces

Absorption Rate of TCDD to the Blood Via the Lymphatic Circulation

Absorption Rate of TCDD by the Liver Via by Portal Circulation


    ACKNOWLEDGMENTS
 
The authors are grateful to Drs Hugh Barton and Barbara Abbott for their excellent comments on initial drafts of this manuscript. This project was funded by in part by a cooperative agreement (CR 828790) with NRC, NAS, and performed at US EPA Research Triangle Park, NC.


    NOTES
 
Disclaimer: This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Approval does not signify that the content necessarily reflects the view and policies of the agency, nor does mention of the trade names or commercial products constitute endorsement or recommendation for use,

1 To whom correspondence should be addressed at U.S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Environmental Toxicology division, Pharmacokinetics Branch, Mail drop B143–01 Research Triangle Park, NC 27711. Fax: (919) 541-4284. E-mail: Devito.Mike{at}epa.gov.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX 1: EQUATIONS FOR...
 REFERENCES
 
Abbott, B. D., Birnbaum, L. S., and Diliberto, J. J. (1996). Rapid distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to embryonic tissues in C57BL/6N mice and correlation with palatal uptake in vitro. Toxicol. Appl. Pharmacol. 141, 256–263.[CrossRef][ISI][Medline]

Abraham, K., Krowke, R., and Neubert, D. (1988). Pharmacokinetics and biological activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. 1. Dose-dependent tissue distribution and induction of hepatic ethoxyresorufin O-deethylase in rats following a single injection. Arch. Toxicol. 62, 359–368.[ISI][Medline]

Andersen, M. E., and Dennison, J. E. (2001). Mode of action and tissue dosimetry in current and future risk assessments. Sci. Total Environ. 274, 3–14.[CrossRef][ISI][Medline]

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 Analy. 13, 25–36.[ISI]

Birnbaum, L. S. (1995). Development effects of dioxins. Environ. Health Perspect. 103, 89–94.

Birnbaum, L. S., and DeVito, M. J. (1995). Use of toxic equivalency factors for risk assessment for dioxins and related compounds. Toxicology 105, 391–401.[CrossRef][ISI][Medline]

Birnbaum, L. S., and Tuomisto, J. (2000). Non-carcinogenic effects of TCDD in animals. Food Addit. Contam. 17, 275–288.[CrossRef][ISI][Medline]

Bjerke, D. L., and Peterson, R. E. (1994). Reproductive toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxine in male rats: Different effects of in utero versus lactational Exposure. Toxicol. Appl. Pharmacol. 127, 214–249.

Borlakoglu, J. T., Scott, A., Henderson, C. J., Jenke, H. J., and Wolf, C. R. (1993). Transplacental transfer of polychlorinated biphenyls induces simultaneously the expression of P450 isoenzymes and the protooncogenes c-Ha-ras and c-raf. Biochem. Pharmacol. 45, 1373–86.[CrossRef][ISI][Medline]

Buelke-Sam, J., Holson, J. F., and Nelson, C. J. (1982a). Blood flow during pregnancy in the rat: II. Dynamics of and litter variability in uterine flow. Teratology 26, 279–288.[ISI][Medline]

Buelke-Sam, J., Nelson,C. J., Byrd, R. A., and Holson, J. F. (1982b). Blood flow during pregnancy in the rat: I. Flow patterns to maternal organs. Teratology 26, 269–277.[ISI][Medline]

Carrier, G. (1991). Réponse de L'Organisme Humain aux BPC, Dioxines et Furannes et Analyse Des Risques Toxiques. Le passeur, Québec.

Carrier, G., Brunet, R. C., and Brodeur, J. (1995a). Modeling of the toxicokinetics of polychlorinated dibenzo-p-dioxins and dibenzofurans in mammalians, including humans. II. Kinetics of absorption and disposition of PCDDs/PCDFs. Toxicol. Appl. Pharmacol. 131, 267–76.[CrossRef][ISI][Medline]

Carrier, G., Brunet, R. C., and Brodeur, J. (1995b). Modeling of the toxicokinetics of polychlorinated dibenzo-p-dioxins and dibenzofurans in mammalians, including humans. I. Nonlinear distribution of PCDD/PCDF body burden between liver and adipose tissues. Toxicol. Appl. Pharmacol. 131, 253–66.[CrossRef][ISI][Medline]

Chen, C. Y., Hamm, J. T., Hass, J. R., Albro, P. W., and Birnbaum, L. S. (2002). A mixture of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and non-ortho polychlorinated biphenyls (PCBs) changed the lipid content of pregnant Long Evans rats. Chemosphere 46, 1501–1504.[CrossRef][ISI][Medline]

Clewell, R. A., Merrill, E. A., Yu, K. O., Mahle, D. A., Sterner, T. R., Mattie, D. R., Robinson, P. J., Fisher, J. W., and Gearhart, J. M. (2003). Predicting fetal perchlorate dose and inhibition of iodide kinetics during Gestation: A physiologically-based pharmacokinetic analysis of perchlorate and iodide kinetics in the rat. Toxicol. Sci. 73, 235–55.[Abstract/Free Full Text]

DeVito, M. J., and Birnbaum, L. S. (1994). Toxicology of dioxins and related chemicals. In Dioxin and Health (A. Schecter, Ed.), pp. 139–162. Plenum Press, New York.

DeVito, M. J., Birnbaum, L. S., Farland, W. H., and Gasiewicz, T. A. (1995). Comparisons of estimated human body burdens of dioxinlike chemicals and TCDD body burdens in experimentally exposed animals. Environ. Health Perspect. 103, 820–831.[ISI][Medline]

DeVito, M. J., Kim, A., Walker, N. J., Parham, F. M., and Portier, C. J. (2003). Dose-response relationships for dioxins. In Dioxins and Health (A. Schecter and T. Gasiewicz, Eds.), pp. 247–299. John Wiley & Sons Inc. Hoboken, NJ.

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, 223–234.[Abstract]

Diliberto, J. J., Burgin, D., and Birnbaum, L. S. (1997). Role of CYP1A2 in hepatic sequestration of dioxin: studies using CYP1A2 knock-out mice. Biochem. Biophys. Res. Commun. 236, 431–433.[CrossRef][ISI][Medline]

Diliberto, J. J., Burgin, D. E., and Birnbaum, L. S. (1999). Effects of CYP1A2 on disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 2,2',4,4',5,5'-hexachlorobiphenyl in CYP1A2 knockout and parental (C57BL/6N and 129/Sv) strains of mice. Toxicol. Appl. Pharmacol. 159, 52–64.[CrossRef][ISI][Medline]

Diliberto, J. J., DeVito, M. J., Ross, D. G., and Birnbaum, L. S. (2001). Subchronic exposure of [3H]- 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in female B6C3F1 mice: Relationship of steady-state levels to disposition and metabolism. Toxicol. Sci. 61, 241–255.[Abstract/Free Full Text]

Faqi, A. S., Dalsenter, P. R., Merker, H. J., and Chahoud, I. (1998). Reproductive toxicity and tissue concentrations of low doses of 2,3,7,8- tetrachlorodibenzo-p-dioxin in male offspring rats exposed throughout pregnancy and lactation. Toxicol. Appl. Pharmacol. 150, 383–392.[CrossRef][ISI][Medline]

Fenton, S. E., Hamm, J. T., Birnbaum, L. S., and Youngblood, G. L. (2002). Persistent abnormalities in the rat mammary gland following gestational and lactational exposure to 2,3,7,8 -tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Sci. 67, 63–74.[Abstract/Free Full Text]

Fisher, J. W., Whittaker, T. A., Taylor, D. H., and Clewell, H. J., III. (1989). Physiologically based pharmacokinetic modeling of the pregnant rat: A multiroute exposure model for trichloroethylene and its metabolite, trichloroacetic acid. Toxicol. Appl. Pharmacol. 99, 395–414.[ISI][Medline]

Gentry, P., Covington, T., Andersen, M., and Clewell, H. J. (2002). Application of a physiologically based pharmacokinetic model for isopropanol in the derivation of a reference dose and reference concentration. Regul. Toxicol. Pharmacol. 36, 51–68.[CrossRef][ISI][Medline]

Gentry, P. R., Covington, T. R., and Clewell, H. J. (2003). Evaluation of the potential impact of pharmacokinetic differences on tissue dosimetry in offspring during pregnancy and lactation. Regul. Toxicol. Pharmacol. 38, 1–16.[CrossRef][ISI][Medline]

Gray, L. E., Kelce, W. R., Monosson, E., Ostby, J. S., and Birnbaum, L. S. (1995). Exposure to TCDD during development permanently alters reproductive function in male Long Evans rats and hamsters: Reduced ejaculated and epididymal sperm numbers and sex accessory gland weights in offspring with normal androgenic status. Toxicol. Appl. Pharmacol. 131, 108–118.[CrossRef][ISI][Medline]

Gray, L. E., Ostby, J. S., and Kelce, W. R. (1997a). A dose-response analysis of the reproductive effects of a single gestational dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male Long Evans Hooded rat offspring. Toxicol. Appl. Pharmacol. 146, 11–20.[CrossRef][ISI][Medline]

Gray, L. E., Wolf, C., Mann, P., and Ostby, J. S. (1997b). In utero exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin alters reproductive development of female Long Evans Hooded rat offspring. Toxicol. Appl. Pharmacol. 146, 237–244.[CrossRef][ISI][Medline]

Hayes, A. W. (1994). Principles and Methods of Toxicology. Raven Press, New York.

Hurst, C. H., Abbott, B., Schmid, J. E., Birnbaum, L. S. (2002). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) disrupts early morphogenetic events that form the lower reproductive tract in female rat fetuses. Toxicol. Sci. 65, 87–98.[Abstract/Free Full Text]

Hurst, C. H., Abbot, B. D., DeVito, M. J., Birnbaum, L. S. (1998). 2,3,7,8-tetrachlorodibenzo-p-dioxin in pregnant Long Evans rats: disposition to maternal and embryo/fetal tissues. Toxicol. Sci. 45, 129–136.[Abstract]

Hurst, C. H., DeVito, M. J., and Birnbaum, L. S. (2000a). Tissue disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in maternal and developing Long-Evans rats following subchronic exposure. Toxicol. Sci. 57, 275–283.[Abstract/Free Full Text]

Hurst, C. H., DeVito, M. J, Setzer, R. W., and Birnbaum, L. S. (2000b). Acute administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in pregnant Long Evans rats: Association of measured tissue concentrations with developmental effects. Toxicol. Sci. 53, 411–420.[Abstract/Free Full Text]

Kohn, M. C., Walker, N. J., Kim, A. H., Portier, C. J. (2001). Physiological modeling of a proposed mechanism of enzyme induction by TCDD. Toxicology 162, 193–208.[CrossRef][ISI][Medline]

Krishnan, K., Haddad, S., and Pelekis, M. (1995). A simple index for representing the discrepancy between simulation of physiological pharmacokinetic models and experimental data. Toxicol. Indust. Health 11, 413–422.[ISI][Medline]

Krowke, R., Chahoud, I., Baumann-Wilschke, I., and Neubert, D. (1989). Pharmacokinetics and biological activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. 2. Pharmacokinetics in rats using a loading-dose/maintenance-dose regime with high doses. Arch. Toxicol. 63, 356–360.[ISI][Medline]

Lawrence, G. S., and Gobas, F. A. (1997). A pharmacokinetic analysis of interspecies extrapolation in dioxin risk assessment. Chemosphere 35, 427–452.[CrossRef][ISI][Medline]

Leung, H. W., Ku, R. H., Paustenbach, D. J., and Andersen, M. E. (1988). A physiologically based pharmacokinetic model for 2,3,7,8-tetrachlorodibenzo-p-dioxin in C57Bl/6 J and DBA/2 J mice. Toxicol. Lett. 42, 15–28.[CrossRef][ISI][Medline]

Leung, H. W., Paustenbach, D. J., Murray, F. J., and Andersen, M. E. (1990). A physiological pharmacokinetic description of the tissue distribution and enzyme-inducing properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Toxicol. Appl. Pharmacol. 103, 399–410.[ISI][Medline]

Li, X., Weber, L. W., and Rozman, K. K. (1995). Toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats including placental and lactational transfer to fetuses and neonates. Fundam. Appl. Toxicol. 27, 70–76.[CrossRef][ISI][Medline]

Lin, T. M., Simanainen, U., Moore, R. W., Peterson, R. E. (2002). Critical windows of vulnerability for effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on prostate and seminal vesicle development in C57BL/6 mice. Toxicol. Sci. 69, 202–9.[Abstract/Free Full Text]

Mably, T. A., Bjerke, D. L., Moore, R. W., Gendron-Fitzpatrick, A., and Peterson, R. E. (1992a). In utero and lactational exposure of male rats to 2,3,7,8- tetrachlorodibenzo-p-dioxin. 3. Effects on spermatogenesis and reproductive capability. Toxicol. Appl. Pharmacol. 114, 118–126.[ISI][Medline]

Mably, T. A., Moore, R. W., Goy, R. W., and Peterson, R. E. (1992b). In utero and lactational exposure of male rats to 2,3,7,8- tetrachlorodibenzo-p-dioxin. 2. Effects on sexual behavior and the regulation of luteinizing hormone secretion in adulthood. Toxicol. Appl. Pharmacol. 114, 108–117.[ISI][Medline]

Mably, T. A., Moore, R. W., and Peterson, R. E. (1992c). In utero and lactational exposure of male rats to 2,3,7,8- tetrachlorodibenzo-p-dioxin. 1. Effects on androgenic status. Toxicol. Appl. Pharmacol. 114, 97–107.[ISI][Medline]

Markowski, V. P., Zareba, G., Stern, S., Cox, C., and Weiss, B. (2001). Altered operant responding for motor reinforcement and the determination of benchmark doses following perinatal exposure to low-level 2,3,7, 8-tetrachlorodibenzo-p-dioxin. Environ. Health Perspect. 109, 621–627.[ISI][Medline]

Maruyama, W., Yoshida, K., Tanaka, T., and Nakanishi, J. (2002). Possible range of dioxin concentration in human tissues: Simulation with a physiologically based model. J. Toxicol. Environ. Health A 65, 2053–2073.[CrossRef][ISI]

Maruyama, W., Yoshida, K., Tanaka, T., Nakanishi, J. (2003). Simulation of dioxin accumulation in human tissues and analysis of reproductive risk. Chemosphere 53, 301–13.[CrossRef][ISI][Medline]

Mattison, D. R., Blann, E., and Malek, A. (1991). Physiological alterations during pregnancy: Impact on toxicokinetics. Fundam. Appl. Toxicol. 16, 215–218.[ISI][Medline]

Nichols, J. W., Jensen, K. M., Tietge, J. E., and Johnson, R. D. (1998). Physiologically based toxicokinetic model for maternal transfer of 2,3,7,8-tetrachlorodibenzo-p-dioxin in brook trout (Salvelinus fontinalis). Environ. Toxicol. Chem. 17, 2422–2434.[ISI]

Nohara, K., Fujimaki, H., Tsukumo, S., Ushio, H., Miyabara, Y., Kijima, M., Tohyama, C., and Yonemoto, J. (2000). The effects of perinatal exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin on immune organs in rats. Toxicology 154, 123–133.[CrossRef][Medline]

O'Flaherty, E. J., Scott, W., Schreiner, C., and Beliles, R. P. (1992). A physiologically based kinetic model of rat and mouse gestation: Disposition of a weak acid. Toxicol. Appl. Pharmacol. 112, 245–256.[ISI][Medline]

O'Flaherty, E. J. (1994). Physiologically based pharmacokinetic models in developmental toxicology. Risk Anal. 14, 605–611.[ISI][Medline]

Oesterheld, J. R. (1998). A review of developmental aspects of cytochrome P450. J. Child Adolesc. Psychopharmacol. 8, 161–174.[ISI][Medline]

Ohsako, S., Miyabara, Y., Nishimura, N., Kurosawa, S., Sakaue, M., Ishimura, R., Sato, M., Takeda, K., Aoki, Y., Sone, Y., et al. (2001). Maternal exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the development of reproductive organs of male rats: Dose-dependent increase of mRNA levels of 5-reductase type 2 in contrast to decrease of androgen receptor in the pubertal ventral prostate. Toxicol. Sci. 60, 132–143.[Abstract/Free Full Text]

Patterson, D. G., Jr., Hampton, L., Lapeza, C. R. J., Belser, W. T., Green, V., Alexander, L., and Needham, L. L. (1987). High-resolution gas chromatographic/high-resolution mass spectrometric analysis of human serum on a whole-weight and lipid basis for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Anal. Chem. 59, 2000–2005.[ISI][Medline]

Poland, A., Teitelbaum, P., and Glover, E. (1989). [125I]2-iodo-3,7,8-trichlorodibenzo-p-dioxin-binding species in mouse liver induced by agonists for the Ah receptor: Characterization and identification. Mol. Pharmacol. 36, 113–120.[Abstract]

Roth, W. L., Freeman, R. A., and Wilson, A. G. (1993). A physiological based model for gastrointestinal absorption and excretion of chemicals carries by lipids. Risk Anal. 13, 531–543.[ISI][Medline]

Santostefano, M. J., Johnson, K. L., Whisnant, N. A., Richardson, V. M., DeVito, M. J., Diliberto, J. J., and Birnbaum, L. S. (1996). Subcellular localization of TCDD differs between the liver, lungs, and kidneys after acute and subchronic exposure: Species/dose comparisons and possible mechanism. Fundam. Appl. Toxicol. 34, 265–275.[CrossRef][ISI][Medline]

Sikov, M. R., and Thomas, J. M. (1970). Prenatal growth of the rat. Growth 34, 1–14.[ISI][Medline]

Sonnier, M., and Cresteil, T. (1998). Delayed ontogenesis of CYP1A2 in the human liver. Eur. J. Biochem. 251, 893–898.[Abstract]

Steiner, E. C., Rey, T. D., and McCroskey, P. S. (1990). Reference guide for Simusolv. The Dow chemical Company, Midland, MI.

Sweeney, L. M., Tyler, T. R., Kirman, C. R., Corley, R. A., Reitz, R. H., Paustenbach, D. J., Holson, J. F., Whorton, M. D., Thompson, K. M., and Gargas, M. L. (2001). Proposed occupational exposure limits for select ethylene glycol ethers using PBPK models and Monte Carlo simulations. Toxicol. Sci. 62, 124–139.[Abstract/Free Full Text]

Theobald, H. M., and Peterson, R. E. (1997). In utero and lactational exposure to 2,3,7,8 -tetrachlorodibenzo-p-dioxin: Effect on development of the male and female reproductive system of the mouse. Toxicol. Appl. Pharmacol. 145, 124–135.[CrossRef][ISI][Medline]

Tucker Blackburn, S., and Lee Loper, D. (1992). The prenatal period and placental physiology. In Maternal, Fetal, and Neonatal Physiology: A Clinical Perspective (S. Tucker Blackburn and D. Lee Loper, Eds.), pp. 36–88. W. B. Saunders Company, Philadelphia, PA.

Van den Berg, M., Heeremans, C., Veenhoven, E., and Olie, K. (1987). Transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans to fetal and neonatal rats. Fundam. Appl. Toxicol. 9, 635–644.[ISI][Medline]

Van Miller, J. P., Marlar, R. J., and Allen, J. R. (1976). Tissue distribution and excretion of tritiated tetrachlorodibenzo-p-dioxin in non-human primates and rats. Food Cosmet. Toxicol. 14, 31–34.[ISI][Medline]

Vorderstrasse, B. A., Fenton, S. E., Bohn, A. A., Cundiff, J. A., and Lawrence, B. P. (2004). A novel effect of dioxin: Exposure during pregnancy severely impairs mammary gland differentiation. Toxicol. Sci. 78, 248–257.[Abstract/Free Full Text]

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, 151–168.[CrossRef][ISI][Medline]

Wang, X., Santostefano, M. J., DeVito, M. J., and Birnbaum, L. S. (2000). Extrapolation of a PBPK model for dioxins across dosage regimen, gender, strain, and species. Toxicol. Sci. 56, 49–60.[Abstract/Free Full Text]

Wilson, J. G. (1965). Embryological considerations in teratology, in Teratology Principles and Techniques (J. G. Wilson and J. Warkany, Eds), pp. 251–277. The University of Chicago Press, Chicago.

Wolf, C. J., Ostby, J. S., and Gray, L. E., Jr. (1999). Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) severely alters reproductive function of female hamster offspring. Toxicol. Sci. 51, 259–64.[Abstract]