Extrapolation of a PBPK model for dioxins across dosage regimen, gender, strain, and species.

A physiologically based pharmacodynamic (PBPK) model for 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was developed based on pharmacokinetic data from acute oral exposures of TCDD to female Sprague-Dawley rats (Wang et al., 1997, Toxicol Appl. Pharmacol 147, 151-168). In the present study, the utility of this model to predict the disposition of TCDD in male and female Sprague-Dawley and female Wistar rats exposed to TCDD through different dosage regimens was examined. The ability of the model to predict the disposition of 2-iodo-3,7,8-trichlorodibenzo-p-dioxin (ITrCDD) in mice (Leung, et al., 1990, Toxicol. Appl. Pharmacol. 103, 399-410) was also examined. The ability of the model to predict across routes of exposure was assessed with intravenous injection data (5.6 microg/kg bw) (Li et al., 1995, Fundam. Appl. Toxicol. 27, 70-76) in female rats. Analysis across gender extrapolations used data for male Sprague-Dawley rats exposed intravenously to 9.25 microg TCDD/kg bw (Weber et al., 1993, Fundam. Appl. Toxicol. 21, 523-534). The analysis of across-dosage regimen and stains of rats extrapolations were assessed using data from rats exposed to TCDD through a loading/maintenance dosage regimen (Krowke et al., 1989, Arch. Toxicol. 63, 356-360). The physiological differences between gender, strain, and species were taken into account when fitting the PBPK model to these data sets. The results demonstrate that the PBPK model for TCDD developed for female Sprague-Dawley rats exposed by acute oral dosing accurately predicts the disposition of TCDD, for different gender and strain of rats across varying dosage regimens, as well as in a strain of mice. Minimal changes in fitted parameters were required to provide accurate predictions of these data sets. This study provides further confirmation of the potential use of physiological modeling in understanding pharmacokinetics and pharmacodynamics.

Dose-dependent localization of TCDD in isolated centrilobular and periportal hepatocytes.

Dose-response relationships for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suggest a differential sensitivity of liver cell types to the induction of cytochrome P450 gene expression, and that the induction of hepatic protein CYP1A2 causes sequestration of TCDD. In addition, immunolocalization of hepatic CYP1A1/1B1/1A2 proteins is not uniform after exposure to TCDD. The mechanism for the regio-specific induction of hepatic P450s by TCDD is unknown, but may involve the differential distribution of participants in the AhR-mediated pathway and/or regional P450 isozymes, as well as, non-uniform distribution/sequestration of TCDD. Therefore, this study examined the effects of TCDD in unfractionated, centrilobular and periportal hepatocytes isolated from female Sprague-Dawley rats acutely exposed (3 days) to a single oral dose of 0.01-10.0 microg [3H]TCDD/kg. A dose-dependent increase in concentration of TCDD was accompanied by a dose-dependent increase in CYP1A1, CYP1A2, and CYP1B1 mRNA expression and associated enzymes in all liver-cell populations. Centrilobular hepatocytes showed a 2.7- to 4.5-fold higher concentration of TCDD as compared to the periportal hepatocytes at doses up to 0.3 microg TCDD/kg. Centrilobular hepatocytes also exhibited an elevated MROD activity as compared to the periportal hepatocytes at doses up to 0.3 microg TCDD/kg. Furthermore, centrilobular hepatocytes showed an elevated concentration of induced CYP1A2 and CYP1B1 mRNA as compared to periportal hepatocytes within the 0.01- and 0.3-microg TCDD/kg-treatment groups. This is the first study to demonstrate that a dose-dependent difference in distribution of TCDD exists between centrilobular and periportal cells that might be related to regional differences in P450 induction.

Daily cycle of bHLH-PAS proteins, Ah receptor and Arnt, in multiple tissues of female Sprague-Dawley rats.

The aryl hydrocarbon receptor (AhR) shares a common PAS domain with a number of genes that exhibit a pronounced circadian rhythm. Therefore, this study examined the daily cycle of AhR and AhR nuclear translocator (Arnt) protein expression in multiple tissues of female Sprague-Dawley rats. Rats were euthanized at 4, 7, and 11 am and 4, 7, and 11 pm after which whole tissue homogenates were made from multiple tissues. Western blot analysis showed that the daily cycle of relative AhR protein expression exhibits a similar oscillation pattern in the liver, lungs, and thymus. The daily cycle of relative Arnt protein expression exhibits a similar oscillation pattern in the liver and lungs. The apparent daily cycle of AhR and Arnt protein expression in multiple tissues was not observed within the spleen. This preliminary report is the first study to suggest that the PAS proteins, AhR and Arnt, exhibit a daily oscillation pattern within multiple target tissues which may give insight into the tissue-specific toxic and biochemical responses mediated through this dimerization pair, as well as the physiological function of these proteins.

A pharmacodynamic analysis of TCDD-induced cytochrome P450 gene expression in multiple tissues: dose- and time-dependent effects.

The ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) to alter gene expression and the demonstration that the induction of CYP1A2 is responsible for hepatic TCDD sequestration suggest that both pharmacokinetic and pharmacodynamic events must be incorporated for a quantitative description of TCDD disposition. In this paper, a biologically based pharmacodynamic (BBPD) model for TCDD-induced biochemical responses in multiple tissues was developed. The parameters responsible for tissue response were estimated simultaneously with a refined physiologically based pharmacokinetic (PBPK) model developed by Wang et al. (1997a), by using the time-dependent effects of TCDD on induced CYP1A1/CYP1A2 gene expression in multiple target tissues (liver, lungs, kidneys, and skin) of female Sprague-Dawley rats treated with 10 microgram TCDD/kg for 30 min, 1, 3, 8, or 24 h, or 7, 14, or 35 days. This refined BBPD model developed based on the time-course of TCDD-induced CYP1A1/CYP1A2 protein expression, and associated enzymatic activities well described the dose-dependent effects of TCDD on cytochrome P450 protein expression and associated enzyme activities in the multiple tissues of female Sprague-Dawley rats at 3 days following a single exposure to TCDD (0.01-30.0 micromgram TCDD/kg). This is the first BBPD model to quantitatively describe the time- and dose-dependent effects of TCDD on induced CYP1A1/CYP1A2 protein expression and associated enzyme activities in multiple target tissues for TCDD-induced biochemical responses.

Female Sprague-Dawley rats exposed to a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibit sustained depletion of aryl hydrocarbon receptor protein in liver, spleen, thymus, and lung.

There is currently little information concerning the time-dependent relationship between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure and aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) protein concentration in vivo. Therefore, female Sprague-Dawley rats were given a single oral dose of TCDD (10 micrograms/kg), and the AHR and ARNT protein concentrations in liver, spleen, thymus, and lung determined by Western blotting. In liver, the concentration of AHR protein was significantly reduced 8 and 24 h postdosing as compared to time-matched controls. In spleen and lung, the concentration of AHR protein was reduced 3, 8, 24, and 168 h posttreatment compared to time-matched controls but returned to control levels by 336 h. In thymus, reductions in AHR protein concentration were observed 8, 24, 168, and 336 h postdosing as compared to time-matched controls. Significant reductions in the concentration of ARNT protein were not observed in any of the TCDD-exposed tissues. Functional studies in cell culture showed that exposure of a mouse hepatoma cell line (Hepa-1c1c7) and a rat smooth muscle cell line (A-7) to TCDD (1 nM) for 12 days resulted in a 50% reduction in TCDD-inducible reporter gene expression following subsequent challenge by an additional dose of TCDD (1 nM). Collectively, these results show that (i) TCDD-mediated depletion of AHR occurs in vivo, (ii) AHR protein does not rapidly recover to pretreatment levels even though the tissue concentration of TCDD has fallen, and (iii) reduction in AHR protein concentration correlates with reduction in TCDD-mediated reporter gene expression in mammalian culture cells.

Determination of parameters responsible for pharmacokinetic behavior of TCDD in female Sprague-Dawley rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic member of a class of planar and halogenated chemicals. Improvements in exposure assessment of TCDD require scientific information on the distribution of TCDD in target tissues and cellular responses induced by TCDD. Since 1980, several physiologically based pharmacokinetic (PBPK) models for TCDD and related compounds have been reported. Some of these models incorporated the induction of a hepatic binding protein in response to interactions of TCDD, the Ah receptor, and DNA binding sites and described the TCDD disposition in a biological system for certain data sets. Due to the limitations of the available experimental data, different values for the same physical parameters of these models were obtained from the different studies. The inconsistencies of the parameter values limit the application of PBPK models to risk assessment. Therefore, further refinement of previous models is necessary. This paper develops an improved PBPK model to describe TCDD disposition in eight target tissues. The interaction of TCDD with the Ah receptor and with hepatic inducible CYP1A2 were also incorporated into the model. This model accurately described the time course distribution of TCDD following a single oral dose of 10 microg/kg, as well as the TCDD concentration on Day 3 after six different doses, 0.01, 0.1, 0.3, 1, 10, and 30 microg TCDD/kg, in target tissues. This study extends previous TCDD models by illustrating the validity and the limitation of the model and providing further confirmation of the potential PBPK model for us in optimal experimental design and extrapolation across doses and routes of exposure. In addition, this study demonstrated some critical issues in PBPK modeling.

Differential time-course and dose-response relationships of TCDD-induced CYP1B1, CYP1A1, and CYP1A2 proteins in rats.

This study examined the relationship between dose- and time-dependent hepatic localization of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and expression of CYP1B1, CYP1A1 and CYP1A2 proteins. A dose-dependent increase in hepatic TCDD in female Sprague-Dawley rats treated with 0.01-30.0 microg TCDD/kg was observed. TCDD induced CYP1A1 protein in rats treated with 0.3 microg TCDD/kg or higher. TCDD induced CYP1A2 and CYP1B1 proteins in rats treated with 1.0 microg TCDD/kg or higher. The in vivo ED50 (microg TCDD/kg) for TCDD-induced CYP1A1, CYP1A2 and CYP1B1 proteins were 0.22, 0.40 and 5.19, respectively. Hepatic accumulation of TCDD reached a maximum at 8 hours post dosing with a t1/2 of approximately 10 days. TCDD-induced CYP1A1/CYP1A2 protein expression was increased time-dependently, reaching a maximum at 3 days after dosing and remaining elevated for 35 days. In contrast, TCDD-induced CYP1B1 protein showed significant expression at 3 days after dosing and decreased to basal concentrations by 35 days. This study demonstrates that TCDD exhibits differential dose-response and time-course relationships on hepatic localization and cytochrome P-450 protein expression.

Subcellular localization of TCDD differs between the liver, lungs, and kidneys after acute and subchronic exposure: species/dose comparisons and possible mechanism.

Subcellular localization of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds has been examined only in the liver. The objective of this study was (1) to examine and compare the subcellular distribution of TCDD within hepatic and nonhepatic (lungs/kidneys) tissues of female Sprague-Dawley rats acutely exposed to TCDD, (2) to analyze species comparisons in the subcellular localization of TCDD in multiple tissues, (3) to investigate the effect of dose on subcellular distribution of TCDD, (4) to analyze the effect of subchronic exposure on the subcellular distribution of TCDD, and (5) to examine one possible mechanism for subcellular localization of TCDD. Female Sprague-Dawley rats and B6C3F1 mice received a single oral dose of 0.1, 1.0, or 10 microg [3H]TCDD/kg body weight and subcellular fractions of the liver, lungs, and kidneys were prepared by differential centrifugation 3 days after exposure. Analysis of the rat subcellular fractions revealed that TCDD was equally distributed between the hepatic P9 (mitochondrial, lysosomal, and nuclear) and S9 (cytosol and microsomal) fractions at all doses tested. In contrast, TCDD was concentrated in the P9 of rat nonhepatic tissues at all doses studied. Differential centrifugation of the hepatic S9 showed that TCDD was localized within the hepatic P100 (microsomal) fraction at all doses tested. In contrast, TCDD localized in pulmonary and renal S100 (cytosolic) fractions at all doses. The subcellular distribution of TCDD in the liver and lungs of acutely exposed B6C3F1 mice was similar to that observed in the rats. Although TCDD was concentrated within the renal P9, the remainder of TCDD in the S9 was evenly distributed between the S100 and the P100 fractions of acutely exposed B6C3F1 mice. Subchronic exposure of B6C3F1 mice to 1.5 or 150 ng [3H]TCDD/kg/day revealed that increasing dose resulted in equal distribution of TCDD between the hepatic S9 and P9 versus concentration in the renal P9. In addition, a dose-dependent increase in accumulation of TCDD in the hepatic P100 was accompanied by a dose-dependent increase in TCDD localization in the renal S100 of mice subchronically exposed to TCDD. TCDD exposure in rats resulted in a dose-dependent increase in the induction of CYP1A1 protein and associated enzyme activity in hepatic, pulmonary, and renal microsomes. TCDD-induced CYP1A2 protein levels and associated enzymatic activity were only present in hepatic microsomes. This is the first report to suggest that subcellular distribution of TCDD differs between hepatic and nonhepatic tissues and demonstrate that the liver-specific microsomal localization of TCDD in female Sprague-Dawley rats also occurs in the liver of female B6C3F1 mice acutely or subchronically exposed to TCDD. In addition, these data are consistent with the hypothesis that the hepatic sequestration of TCDD is due to an interaction with CYP1A2. Furthermore, the lack of pulmonary/renal sequestration coupled with the lack of localization of TCDD in pulmonary/renal microsomes also supports the role of CYP1A2 as a hepatic microsomal binding protein involved in TCDD sequestration..