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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.

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.

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., (Copyright 1997 Academic Press.)

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..

The potential non-additive interactions of polynuclear aromatic hydrocarbon (PAH) mixtures as inducers of Cyp1a-1 and Cyp1a-2 gene expression were investigated in B6C3F1 mice using a reconstituted PAH mixture. The chemical composition (% by weight) of the reconstituted PAH mixture was: 2-ring PAHs--indan (0.22), naphthalene (23.8), 2-methylnaphthalene (23.2) and 1-methylnaphthalene (13.3); 3-ring PAHs--acenaphthylene (7.7), acenaphthene (0.6), dibenzofuran (0.7), fluorene (4.3), phenanthrene (10.5) and anthracene (3.4); > or = 4-ring PAHs--fluoranthene (2.4), pyrene (4.3), benz[a]anthracene (1.4), chrysene (1.5), benzo[b]fluoranthene (0.8), benzo[k]fluoranthene (0.9) and benzo[a]pyrene (0.9). The composition of the 2-, 3- and > or = 4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The > or = 4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The > or = 4-ring PAH fraction and reconstituted mixture induced hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity and Cyp1a-1 mRNA levels, whereas the 2- and 3-ring PAHs were only weakly active. Direct comparison of the potencies of the reconstituted mixture and > or = 4-ring PAHs showed that the Cyp1a-1 induction activity of the reconstituted mixture was due to the > or = 4-ring PAHs. The reconstituted PAH mixture and > or = 4-ring PAHs also induced Cyp1a-2 hepatic mRNA levels and microsomal methoxyresorufin O-deethylase (MROD) activity; however, their dose-response curves indicated that the reconstituted PAH mixture was more potent as a Cyp1a-2 inducer than the > or = 4 ring PAHs. The differences in potency were due to 3-ring PAHs which were found to be strong inducers of hepatic Cyp1a-2 mRNA levels and microsomal MROD activity at the lowest dose administered (37 mg/kg). The 3-ring mixture caused a maximal 29-fold increase in hepatic MROD activity at a dose of 292 mg/kg, but only 28% of maximal induction of EROD activity. Northern analysis of liver mRNA from mice treated with 3-ring PAHs showed that there was minimal induction of Cyp1a-1 mRNA levels. The 3-ring PAHs did not competitively bind to the mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor suggesting that 3-ring PAHs are a new class of Cyp1a-2 inducers which do not act through the Ah receptor.

The competitive binding of 3'-methoxy-4'-nitro, 4'-amino-3'-methoxy, 4'-methoxy-3'-nitro, and 3'-amino-4'-methoxyflavone (compounds 1 to 4, respectively) to the rat cytosolic aryl hydrocarbon (Ah) receptor gave IC50 values of 2.27, 86.1, 872, and 19.4 nM. Flavones 3 and 4 were characterized as Ah receptor agonists in MCF-7 human breast cancer cells and induced CYP1A1 gene expression, whereas the 3-methoxy-substituted flavones (1 and 2) were inactive. All four compounds inhibited induction of ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells; moreover, in vitro studies with TCDD-induced rat liver microsomes showed that flavones 1 to 4 inhibited EROD activity in the presence or absence of NADPH. In MCF-7 cells cotreated with flavones 1 or 2 (0.01 to 10 microM) plus TCDD or [3H]TCDD, there was a concentration-dependent inhibition of TCDD-induced CYP1A1 mRNA levels and formation of radiolabeled nuclear Ah receptor complex. Velocity sedimentation analysis of nuclear extracts from MCF-7 cells treated with [3H]TCDD plus 1 or 10 microM concentrations of flavones 1 and 2 showed that an early eluting specifically bound nuclear Ah receptor complex was present. However, under these same conditions the flavones inhibited TCDD-induced CYP1A1 gene expression. The apparent molecular mass of this nuclear complex was 190 kDa as determined by cross-linking to a 32P-labeled bromodeoxyuridine-substituted consensus dioxin-responsive element. Similar cross-linking results were obtained using the nuclear extract from MCF-7 cells treated with [3H]TCDD alone. The results of this study suggest that there are at least two forms of the nuclear Ah receptor complex in MCF-7 cells; the major transcriptionally active form binds [3H]TCDD and flavones 1 or 2 inhibit nuclear uptake of this receptor complex. The other form of the nuclear Ah receptor complex appears to be transcriptionally inactive and ligand binding with [3H]TCDD is not competitively inhibited by flavones 1 and 2.

The effects of several 2-substituted phenanthridinones (2-nitro-, 2-t-butyl-, 2-bromo-, 2-phenyl-, 2-ethyl-, 2-methoxy-, 2-iodo-, 2-n-butyl-, 2-chloro-, 2-trifluoromethyl-, 2-fluoro-, 2-isopropyl-, and 2-methyl) and phenanthridinone as ligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor were determined using a competitive binding assay and 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. The competitive binding IC50 values varied from 317 (2-trifluoromethyl-) to 5870 nM (2-methoxyphenanthridinone); the relative low Ah receptor binding affinities for these compounds were paralleled by their weak activity as inducers of ethoxyresorufin O-deethylase (EROD) activity in rat hepatoma H4IIE cells; however, there was not a correlation between their structure-binding and structure-induction relationship. In cells cotreated with 1 nM TCDD plus different concentrations (0.01-10 microM) of the 2-substituted phenanthridinones, several of these compounds inhibited induction of EROD activity by TCDD; 2-t-butyl- and 2-phenylphenanthridinone (2-PP) were the most active compounds, causing a > 80% reduction in the induced response. 2-PP was selected as a model to further investigate the mechanism of this inhibitory response. The results of interactive studies in rat hepatoma H4IIE cells cotreated with 2-PP plus TCDD or [3H]TCDD showed that 2-PP did not inhibit formation of the nuclear Ah receptor complex or induction of CYP1A1 mRNA levels or CYP1A1 protein. In contrast, incubation of 2-PP with either rat hepatoma H4IIE cells treated with TCDD or hepatic microsomes from TCDD-treated rats resulted in a rapid loss of EROD activity. In parallel experiments, [3H]2-PP was incubated with hepatic microsomes from TCDD-treated rats and analysis by denaturing electrophoresis showed that [3H]2-PP formed a covalent adduct with a 50- to 55-kDa protein and thus acted as a suicide inactivator of CYP1A1.

A new synthetic route was utilized to prepare 6-substituted 3,4-benzocoumarins where the substituents were iodo, fluoro, trifluoromethyl, bromo, chloro, isopropyl, ethyl, t-butyl, methyl, hydrogen, amino, phenyl, or nitro; 3,4-naphthocoumarin was also synthesized. The relative affinities of these congeners for the aryl hydrocarbon (Ah) receptor were determined using rat hepatic cytosol and 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin ([3H]TCDD) as the radioligand. In addition, the Ah receptor agonist activity of the 6-substituted 3,4-benzocoumarins was determined from their concentration-dependent induction of ethoxyresorufin O-deethylase (EROD) activity. In contrast with many other structural classes of halogenated aromatics, there was not a correlation between the structure-binding versus structure induction relationships for the 6-substituted 3,4-benzocoumarins. These results suggested that some of these congeners may exhibit partial Ah receptor antagonist activities and this was investigated by determining the inhibitory effects of 6-substituted 3,4-benzocoumarins on TCDD-induced EROD activity in rat hepatoma H4II E cells in culture. Only four compounds (6-isopropyl, 6-phenyl, 6-fluoro, and 6-t-butyl) inhibited the TCDD-induced response (21.7 to 64.4% inhibition) and the mechanism of action of the most active inhibitor, 6-t-butyl-3,4-benzocoumarin, was further investigated. In contrast, with other partial Ah receptor antagonists such as alpha-naphthoflavone, cotreatment of rat hepatoma H4II E cells with 1 nM TCDD plus 1 and 10 microM 6-t-butyl-3,4-benzocoumarin did not result in decreased levels of the Ah receptor complex (liganded with TCDD). In addition, there was not significant inhibition of TCDD-induced CYP1A1 mRNA levels or protein as determined by Northern and Western blot analyses. The results suggest that 6-t-butyl-3,4-benzocoumarin or one of its metabolites is a post-translational inhibitor of CYP1A1-dependent enzyme (EROD) activity in this cell line and thus represents a novel Ah receptor-independent inhibition of CYP1A1.

Incubation of radiolabeled, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF),1,2,3,7,8-pentachlorodibenzo-p-dioxin(PeCDD), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), 1,2,7,8-TCDF, and 2,3,7-trichlorodibenzo-p-dioxin (TrCDD) with rat hepatic cytosol for 2 h at 0 degrees C gave liganded aryl hydrocarbon (Ah) receptor complexes which were indistinguishable as determined by velocity sedimentation analysis and DNA-Sepharose column chromatography. Incubation of the cytosol plus the different radioligands for 2 h at 20 degrees C resulted in the formation of Ah receptor complexes which exhibited increased retention times on DNA-Sepharose columns. It was apparent that the amount of specifically bound Ah receptor complex or the levels of the transformed Ah receptor complex which eluted from the column with 0.2-0.3 M salt were dependent on the structure of the radioligand. For example, after incubation for 2 h at 20 degrees C the overall yields of the specifically bound transformed Ah receptor complex were 3.4, 2.0, 1.2, 1.9, 0.3, and 0.1%, respectively, using 2,3,7,8-TCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDD, 1,2,3,7,8-PeCDF, 1,2,7,8-TCDF, and 2,3,7,8-TrCDD as radioligands. A more quantitative measure of the structure-dependent transformation of the liganded cytosolic Ah receptor complex was determined using a gel retardation assay with a consensus synthetic dioxin-responsive element (DRE) (26-mer, duplex). The EC50 values obtained for the concentration-dependent formation of the retarded DRE-Ah receptor complex using 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2,3,7-TrCDD, and 1,2,7,8-TCDF as ligands were 0.26, 0.35, 0.78, 1.75, 27.0, and 220 nM, respectively. The structure-dependent differences in these values were similar to their different potencies as Ah receptor agonists and these data suggest that the structure-dependent transformation of the liganded cytosolic Ah receptor may significantly contribute to the structure-activity relationships observed for 2,3,7,8-TCDD and related compounds.