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The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with constitutive activities and those induced by xenobiotic ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). One unexplained cellular role for the AHR is its ability to promote cell cycle progression in the absence of exogenous ligands, whereas treatment with exogenous ligands induces cell cycle arrest. Within the cell cycle, progression from G(1) to S phase is controlled by sequential phosphorylation of the retinoblastoma protein (RB1) by cyclin D-cyclin-dependent kinase (CDK) 4/6 complexes. In this study, the functional interactions between the AHR, CDK4, and cyclin D1 (CCND1) were investigated as a potential mechanism for the cell cycle regulation by the AHR. Time course cell cycle and molecular experiments were performed in human breast cancer cells. The results demonstrated that the AHR and CDK4 interact within the cell cycle, and the interaction was disrupted upon TCDD treatment. The disruption was temporally correlated with G(1) cell cycle arrest and decreased phosphorylation of RB1. Biochemical reconstitution assays using in vitro-translated protein recapitulated the AHR and CDK4 interaction and showed that CCND1 was also part of the complex. In vitro assays for CDK4 kinase activity demonstrated that RB1 phosphorylation by the AHR/CDK4/CCND1 complex was reduced in the presence of TCDD. The results suggest that the AHR interacts in a complex with CDK4 and CCND1 in the absence of exogenous ligands to facilitate cell cycle progression. This interaction is disrupted by exogenous ligands, such as TCDD, to induce G(1) cell cycle arrest.

The current statistics associated with breast cancer continue to show a relatively high recurrence rate together with a poor survival for aggressive metastatic disease. These findings reflect, in part, the pharmaceutical intractability of processes involved in the metastatic process and highlight the need to identify additional drug targets for the treatment of late-stage disease. In the current study, we report that ligand activation of the aryl-hydrocarbon receptor (AhR) inhibits multiple aspects of the metastatic process in a panel of breast cancer cell lines that represent the major breast cancer subtypes. Specifically, it was observed that treatment with exogenous AhR agonists significantly inhibited cell invasiveness and motility in the Boyden chamber assay and inhibited colony formation in soft agar regardless of estrogen receptor (ER), progesterone receptor, or human epidermal growth factor receptor 2 status. Knockdown of the AhR using small interfering RNA duplexes demonstrated that the inhibition of invasiveness was receptor dependent and that endogenous receptor activity was protective in each cell type examined. The inhibition of invasiveness and anchorage-independent growth correlated with the ability of exogenous AhR agonists to promote differentiation. Finally, exogenous AhR agonists were able to promote differentiation in a putative mammary cancer stem cell line. Cumulatively, these results suggest that the AhR plays an important role in mammary epithelial differentiation and, as such, represent a promising therapeutic target for a range of phenotypically distinct human breast cancers.

Cytochrome P450 CYP1B1 is a relatively recently identified member of the CYP1 gene family. The purpose of this commentary is to review the regulatory mechanisms, metabolic specificity, and tissue-specific expression of this cytochrome P450 and to highlight its unique properties. The regulation of CYP1B1 involves a variety of both transcriptional and post-transcriptional mechanisms. CYP1B1 can metabolize a range of toxic and carcinogenic chemicals in vitro but in some cases with a unique stereoselectivity. Estradiol 4-hydroxylation appears to be a characteristic reaction catalyzed by human CYP1B1. However, there are considerable species differences regarding the regulation, metabolic specificity, and tissue-specific expression of this P450. In humans CYP1B1 is overexpressed in tumor cells, and this has important implications for tumor development and progression and the development of anticancer drugs specifically activated by CYP1B1.

The cytochrome P450 1B1 gene (CYP1B1) is expressed constitutively and is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the human breast adenocarcinoma cell line MCF-7 but not in the human hepatoma cell line HepG2. Genomic DNA isolated from both cell lines was digested with the methylation-sensitive restriction enzyme isoschizomers MspI and HpaII, and subjected to Southern analysis with a probe for the CYP1B1 promoter/enhancer region. Although differences were observed in methylation patterns for the CYP1B1 gene from MCF-7 and HepG2 cells, treatment with the demethylating agent 5-azacytidine (10 microM for 6 days) did not activate CYP1B1 mRNA expression in HepG2 cells. Furthermore, treatment with the histone deacetylase inhibitor trichostatin A (100 nM for 24 h) did not activate CYP1B1 mRNA expression in HepG2 cells. Comparative analysis of the constitutive expression of luciferase/1B1 reporter constructs containing a series of deletions in the 5' enhancer region indicated that in MCF-7 cells the region from -987 to -732 (relative to the transcription start site) was necessary for maximal levels of activity. Mutation of the aryl hydrocarbon receptor response elements (dioxin response elements) in this region showed that the dioxin response elements located at -833 is essential for constitutive gene expression in MCF-7 cells. In HepG2 cells, reporter gene activity was at least equal or greater than the activity observed in MCF-7 cells, which is in marked contrast to the expression of the native CYP1B1 gene. Taken together these findings indicate that the observed cell-specific differences in CYP1B1 constitutive expression are not mediated by DNA promoter/enhancer methylation, but are likely due to either 1) inaccessibility of the 5'-enhancer region in HepG2 cells to transcriptional activators due to a higher order chromatin structure that does not involve histone acetylation, or 2) the action of a repressor protein at cis-elements located outside of the -2296 to +25 region examined with the CYP1B1 reporter constructs. Furthermore, at least one of the dioxin response elements in the enhancer region is required for constitutive expression of CYP1B1.

The AH receptor (AHR) is a ligand-activated transcription factor and member of a growing family of homologous proteins implicated in development. In this study we have characterized the actions of 2,3, 7,8-tetrachlorodibenzofuran (TCDF), a well-studied AHR ligand, and the expression of AHR and selected AHR signal transduction pathway genes in the developing mouse mammary gland. High levels of AHR protein were observed in the mammary glands of C57Bl/6J (AHR +/+) mice during estrous-stimulated growth and branching of terminal end buds (TEBs). Comparative analysis of mammary gland development in AHR -/- and +/+ littermates revealed a 50% reduction in TEBs and an increase in blunt-ended terminal ducts in the AHR null animals. Treatment of mammary glands, removed from estrogen/progesterone-primed C57Bl/6J mice and maintained in organ culture, with TCDF suppressed lobule development (greater than twofold decreases in lobule number and size), with a concomitant suppression of DNA synthesis, as judged by a 35 to 45% decrease in [3H]thymidine incorporation in the TEBs. Immunohistochemical staining patterns for AHR, aryl hydrocarbon nuclear translocator (ARNT; the heterodimerization partner of AHR), and two AHR-regulated genes, Cyp1A1 and Cyp1B1, were similar and not altered by treatment of mammary glands in organ culture with TCDF. The observed differences in the development of mammary glands from AHR +/+ and -/- mice, associated expression of the AHR protein with hormone-dependent lobule development, and suppressive actions of TCDF support the position that, in C57Bl/6J mice, development of the mammary gland is at least in part AHR dependent. Development occurs in the absence of exogenous AHR ligand, suggesting that the unoccupied receptor may function to support the proliferative stages required for full lobule development., (Copyright 1998 Academic Press.)

The implementation of a rat hepatocyte model system and differential display-polymerase chain reaction resulted in the isolation of ZFP-37 as a peroxisome proliferator-responsive gene. In addition to being responsive to peroxisome proliferators, rat ZFP-37 (rZFP-37) mRNA accumulates rapidly after treating cells with several other hepatic tumor promoters, serum, and cycloheximide, indicating that this gene belongs to the immediate-early growth responsive gene family. Although rZFP-37 and mouse ZFP-37 (mZFP-37) are both members of the Krüppel-associated box and C2H2 zinc finger superfamily of proteins, there are several features that distinguish the two proteins. The primary protein sequences of rat and mouse ZFP-37 are highly conserved, especially within the region encoding the 12 C2H2 zinc finger motifs; however, a region believed to be involved in DNA binding in mZFP-37 is divergent in rZFP-37. Mouse ZFP-37 mRNA is expressed almost exclusively in testes and brain, whereas rZFP-37 mRNA is expressed in testes, brain, kidney, spleen, thymus, lung, and at low levels in liver. A major difference between regulation of ZFP-37 in the two species exists as rZFP-37 is induced, while mZFP-37 is repressed, in liver by the administration of the potent peroxisome proliferator Wy 14,643. Despite the fact that mZFP-37 is believed to be important in cell growth and differentiation in testes and brain, the pronounced differences in regulation of this gene in two closely related species preclude an extrapolation to rZFP-37's biological role. Nonetheless, the effects of tumor promoters and mitogens on its expression and the inclusion of rZFP-37 into the immediate-early growth gene families raise the possibility that this gene plays a role in hepatocyte proliferation and/or differentiation., (Copyright 1998 Academic Press.)

Chinese hamster V79 cell lines were constructed for stable expression of human cytochrome P450 1B1 (P450 1B1) in order to study its role in the metabolic activation of chemicals and toxicological consequences. The new V79 cell lines were applied to studies on DNA adduct formation of the polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P). This compound has been found to be an environmental pollutant, and in rodent bioassays it is the most carcinogenic PAH yet discovered. Activation of DB[a,l]P in various metabolizing systems occurs via fjord region DB[a,l]P-11, 12-dihydrodiol 13,14-epoxides (DB[a,l]PDE): we found that DB[a,l]P is stereoselectively metabolized in human mammary carcinoma MCF-7 cells to the (-)-anti- and (+)-syn-DB[a,l]PDE which both bind extensively to cellular DNA. To follow up this study and to relate specific DNA adducts to activation by individual P450 isoforms, the newly established V79 cells stably expressing human P450 1B1 were compared with those expressing human P450 1A1. DNA adduct formation in both V79 cell lines differed distinctively after incubation with DB[a,l]P or its enantiomeric 11,12-dihydrodiols. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts as well as several highly polar DNA adducts as yet unidentified. The proportion of these highly polar adducts to DB[a,l]PDE adducts was dependent upon both the concentration of DB[a,l]P and the time of exposure. In contrast, V79 cells stably expressing human P450 1B1 generated exclusively DB[a,l]PDE-DNA adducts. Differences in the total level of DNA binding were also observed. Exposure to 0.1 microM DB[a,l]P for 6 h caused a significantly higher level of DNA adducts in V79 cells stably expressing human P450 1B1 (370 pmol/mg of DNA) compared to those with human P450 1A1 (35 pmol/mg of DNA). A 4-fold higher extent of DNA binding was catalyzed by human P450 1B1 (506 pmol/mg of DNA) compared to human P450 1A1 (130 pmol/mg of DNA) 6 h after treatment with 0.05 microM (-)-(11R,12R)-dihydrodiol. In cells stably expressing human P450 1B1 the DNA adducts were derived exclusively from the (-)-anti-DB[a,l]PDE. These results indicate that human P450 1B1 and P450 1A1 differ in their regio- and stereochemical selectivity of activation of DB[a,l]P with P450 1B1 forming a higher proportion of the highly carcinogenic (-)-anti-(11R, 12S,13S,14R)-DB[a,l]PDE metabolite.

Our laboratory has cloned the cDNA (Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y.-Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W. , and Greenlee, W. F. (1994) J. Biol. Chem. 269, 13092-13099) and gene (Tang, Y. M., Wo, Y.-Y. P., Jabs, E. W., Stewart, J. C., Sutter, T. R., and Greenlee, W. F. (1996) J. Biol. Chem. 271, 28324-28330) for human CYP1B1, a new member of the cytochrome P450 superfamily. Here, we report on the mapping and function of the CYP1B1 promoter. The CYP1B1 promoter is fully functional, when it is uncoupled from upstream enhancer elements. Deletion analysis and site-directed mutagenesis identified four regulatory elements required for maximum promoter activity: two antisense Sp1 sites (-84 to -89 and -68 to -73), a TATA-like box (-34 to -29), and an initiator motif (-5 to +3). The initiator and the TATA-like elements are both required for basal promoter activity, with enhanced activity mediated by the two antisense Sp1 elements. The CYP1B1 initiator was demonstrated by in vitro transcription analysis to be a positioning element that maintained fidelity of transcription from a single site. Specific binding to a CYP1B1 initiator probe by human nuclear extract proteins was competed either by the highly homologous murine terminal deoxynucleotidyl transferase initiator or, to a lesser extent, by the adenovirus major late initiator. Taken together, these results indicate that the structure and function of the CYP1B1 promoter confers constitutive expression of the gene and assures fidelity of transcription initiation from a single site. The CYP1B1 promoter is distinct from the promoters of the closely related cytochrome P450s CYP1A1 and CYP1A2 and is structurally and functionally similar to the promoters of constitutively expressed genes and at least two viruses.

Cytochrome P450 CYP1B1 is a recently cloned dioxin-inducible form of the cytochrome P450 family of xenobiotic metabolizing enzymes. An antibody raised against a peptide specific for CYP1B1 was found to recognize CYP1B1 expressed in human lymphoblastoid cells but not to recognize other forms of cytochrome P450, particularly CYP1A1 and CYP1A2. Using this antibody, the cellular distribution and localization of CYP1B1 were investigated by immunohistochemistry in a range of malignant tumors and corresponding normal tissues. CYP1B1 was found to be expressed at a high frequency in a wide range of human cancers of different histogenetic types, including cancers of the breast, colon, lung, esophagus, skin, lymph node, brain, and testis. There was no detectable immunostaining for CYP1B1 in normal tissues. These results provide the basis for the development of novel methods of cancer diagnosis based on the identification of CYP1B1 in tumor cells and the development of anticancer drugs that are selectively activated in tumors by CYP1B1.

The environmental contaminant benzo[c]phenanthrene (B[c]Ph) has weak carcinogenic activity in rodent bioassays; however, the fjord region diol epoxides of B[c]Ph, B[c]Ph-3,4-diol 1,2-epoxides (B[c]PhDE), are potent carcinogens. To determine the role of cytochrome P450 isozymes in the activation of B[c]Ph in MCF-7 cells and the low activation of B[c]Ph in mouse skin, cells of the MCF-7 and the human hepatoma HepG2 cell lines were treated with the potent Ah receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prior to exposure to B[c]Ph for 24 h. Mice were treated topically with 1 microg of TCDD or vehicle (control) for 73 h and then with 2 micromol of B[c]Ph for 24 h. In MCF-7 cells, TCDD exposure increased B[c]PhDE-DNA adduct levels more than 3-fold with a 10-fold increase in the (-)-B[c]PhDE-2-dA(t) adduct. Treatment of HepG2 cells with TCDD prior to B[c]Ph application did not increase B[c]PhDE-DNA binding. Total B[c]PhDE-DNA adducts increased 3-fold in TCDD-treated mouse epidermis: the majority of the increase resulted from (+)-B[c]PhDE-1-dA adducts. Analysis of P450 enzymes by Western blotting detected a large increase of P4501B1 but almost no increase in P4501A1 in MCF-7 cells exposed to 10 microM B[c]Ph for 24 or 48 h. In HepG2 cells, there were no detectable levels of P4501A1 or P4501B1 after treatment with 10 microM B[c]Ph for 24 h. In contrast, topical application of 2 micromol of B[c]Ph to mouse skin for 48 or 72 h increased P4501A1, but no P4501B1 was detected. As a measure of P450 activity, the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) was analyzed in microsomes prepared from MCF-7 and HepG2 cells exposed to 0.1% DMSO, 10 microM B[c]Ph, or 10 nM TCDD for 24 or 48 h and from mouse epidermis treated with 1 microg of TCDD, or vehicle control for 72 h, or 2 micromol of B[c]Ph for 48 h. The levels of DMBA metabolites were low or undetectable in microsomes from B[c]Ph-treated MCF-7 and HepG2 cells, but a metabolite pattern consistent with P4501A1 metabolism of DMBA was present in B[c]Ph-exposed mouse epidermal microsomes. TCDD-treated MCF-7 cells, HepG2 cells, and mouse epidermis had DMBA metabolism patterns characteristic of P4501A1 activity. Microsomes from TCDD-treated human cells formed a higher proportion of the proximate carcinogenic metabolite DMBA-3,4-dihydrodiol (16% of total identified metabolites) than TCDD-treated mouse epidermis (2%). In mouse epidermis, the weak ability of B[c]Ph to increase hydrocarbon-metabolizing activity and the increase in mainly P4501A1, leading to formation of the less carcinogenic stereoisomer B[c]PhDE-1, may explain the low carcinogenic activity of B[c]Ph. In a human mammary carcinoma cell line, treatment with B[c]Ph increases mainly P4501B1 and results in formation of a higher proportion of the more carcinogenic B[c]PhDE-2. This indicates that cells in which B[c]Ph treatment increases P4501B1 levels effectively activate B[c]Ph to potent carcinogenic metabolites.

In this report, we present a characterization of the cell-specific expression of two human cytochrome P450 genes, CYP1A1 and CYP1B1, by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The TCDD-dependent induction of CYP1A1 has been studied extensively and serves as the prototype response for a TCDD-signaling pathway initiated by the reversible binding of TCDD to an intracellular receptor [designated the aryl hydrocarbon (Ah) receptor]. CYP1A1 is induced by TCDD to high levels (45-fold increase) in the human hepatoblastoma line HepG2 as compared with the human renal adenocarcinoma line ACHN. In contrast, CYP1B1 is induced selectively in ACHN cells. Cell-specific induction of CYP1A1 and CYP1B1 mRNA correlates with comparable changes in the corresponding proteins and results, at least in part, from transcriptional activation. Characterization of the mechanism(s) for the differential regulation of CYP1A1 was carried out. Nuclear extracts obtained from either cell line following treatment with TCDD displayed equivalent binding to oligonucleotide probes for two dioxin-responsive elements located 5'-ward of the CYP1A1 promoter. This result obtained with broken cell fractions was confirmed by an intact cell DNA protection assay. Possible involvement of negative regulators is suggested by the presence of a negative regulatory element in the 5' flanking region of the CYP1A1 gene and the observed superinduction of CYP1A1 mRNA by cycloheximide in TCDD-treated HepG2 cells. Electromobility shift analysis using negative regulatory element probes, however, did not detect quantitative differences in the binding of nuclear extract proteins obtained from either HepG2 or ACHN cells treated with TCDD. These findings indicate that the ligand-dependent activation and dioxin-responsive element binding of the Ah receptor required for CYP1A1 induction in HepG2 cells also can occur in ACHN cells. We conclude that the repression of TCDD-dependent CYP1A1 induction in ACHN cells occurs at the level of transactivation in the Ah receptor signal transduction pathway.

Previously, we identified a novel human cytochrome P450 cDNA that is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and represents the first member of a new subfamily designated cytochrome P4501B1 (CYP1B1; Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W., and Greenlee, W. F. (1994) J. Biol. Chem. 269, 13092-13099). Here, we report on the isolation and initial characterization of the CYP1B1 gene. The CYP1B1 gene maps to human chromosome 2 at 2p21-22 and contains three exons and two introns. The putative open reading frame starts in the second exon and is 1629 base pairs in length. Southern analysis using DNA probes directed to each of the three exons confirmed that CYP1B1 is a single copy gene. Human CYP1B1 differs from its two most closely related members of the cytochrome P450 superfamily, CYP1A1 and CYP1A2, in the number of exons (3 versus 7) and chromosome location (2 versus 15). A single transcription initiation site was identified by primer extension analysis and S1 nuclease mapping. Based on nucleotide sequence analysis, the CYP1B1 gene lacks a consensus TATA box in the promoter region and contains nine TCDD-responsive enhancer core binding motifs (5'-GCGTG-3') located within a 2.5-kilobase pair genomic fragment 5'-ward of the transcription initiation start site. Deletion analysis of chloramphenicol acetyltransferase reporter gene constructs containing 5' CYP1B1 genomic fragments indicates that a region from -1022 to -835 containing three of the nine core binding motifs contributes to the TCDD-inducible expression of CYP1B1.

Treatment of Sprague-Dawley (SD) rats with a dosing regimen of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) maintaining a steady-state liver concentration of 150 ng/g results in enhanced hepatocyte proliferation in the periportal region, but reduced proliferation in the remainder of the hepatic lobule (Fox et al. (1993) Cancer Res., 53, 2265-2271). Here, we report an initial characterization of the actions of TCDD on hepatocyte proliferation by monitoring DNA synthesis in primary hepatocytes isolated from SD rats. TCDD caused a dose-dependent inhibition (EC50 = 10 pM) of DNA synthesis in primary hepatocytes isolated from either male or female SD rats in the presence or absence of known hepatocyte mitogens (epidermal growth factor, hepatocyte growth factor, and transforming growth factor alpha). No change in DNA synthesis was observed at TCDD concentrations less than 1 pM. Initial characterization of the EGF response system in these cells revealed that TCDD did not alter the specific binding of EGF, or the levels of EGF receptor protein measured in intact cells or cell lysates. TCDD-dependent inhibition of DNA synthesis occurred independently of the suppression observed with transforming growth factor-beta 1. Estradiol did not alter DNA synthesis in the presence or absence of TCDD. Taken together, these findings indicate that TCDD suppresses DNA synthesis via a novel pathway that is non-responsive to estradiol, independent of TGF-beta, and does not involve a decreased ability of hepatocytes to recognize (bind) EGF, a prototype mitogen.

The expression of CYP1B1 has been identified in breast cancer using the reverse transcriptase-polymerase chain reaction and immunoblotting. CYP1B1 mRNA was expressed in the majority of breast tumours and immunoblotting of breast tumours identified a single protein band of molecular weight 60 kDa corresponding to the predicted molecular weight of human CYP1B1. This is the first study to identify CYP1B1 expression in a tumour where it may represent a previously unknown pathway for the metabolism of oestradiol and chemotherapeutic drugs.

Mechanistic studies with phenobarbital (PB), 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) and other liver tumor promoters support a general model of promotion involving negative selection where specifically-mutated cells derive a growth advantage in the presence of persistent mitosuppression. Exposure to these liver tumor promoters appears to transiently enhance hepatocyte replication, presumably via transcriptional activation of growth regulatory genes, leading to a homeostatic increase in mitoinhibitory growth factors in the liver to constrain proliferation. Transforming growth factor beta 1 (TGF-beta), a potent mitoinhibitory growth factor for hepatocytes, has been associated with the mitosuppression caused by PB and certain peroxisomal proliferators. Escape from TGF-beta mitosuppression may involve loss or alteration of function of the mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptor, which is required for TGF-beta 1 activation, or alterations of the TGF-beta types I, II and III signal transduction receptors. A risk assessment based on a negative selection mechanism could be conducted for tumor promotion endpoints with TCDD and compared with current approaches that implicitly regard TCDD as an initiator. Benchmark dose calculation using centrilobular induction of cytochromes P450 1A1 and 1A2 as a surrogate for periportal growth stimulation would provide a rational starting point for application of conventional safety factor approaches, similar to those used with non-cancer effects. In the future, tissue and plasma concentrations of specific growth factors, e.g. TGF-beta or hepatocyte growth factor, HGF, might be considered as more direct dose surrogates for tumor-promoting effects of xenobiotics. Uncertainty factor adjustments to a TCDD benchmark dose calculation should eventually rely on direct knowledge of regulation of specific growth regulatory genes and their receptors in relevant species and on species differences in TCDD pharmacokinetics, instead of application of default animal-to-human and interindividual uncertainty factors.

Previously, levels of a novel human mRNA, detected by a recombinant cDNA designated clone 1, were shown to be increased 50-fold in response to treatment of a keratinocyte cell line with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in part as a function of increased rates of gene transcription (Sutter, T.R., Guzman, K., Dold, K.M., and Greenlee, W.F. (1991) Science 254, 415-418). Here we report the complete corresponding 5.1-kilobase cDNA sequence. A single open reading frame that predicts a protein of 543 amino acid residues was determined by computer-assisted analysis of the cDNA sequence. This predicted protein identifies a new gene subfamily of cytochrome P450, cytochrome P4501B1 (CYP1B1), that maps to human chromosome 2. Southern blot analysis of genomic DNA indicates that the human CYP1B subfamily is likely to contain only this single gene. Northern blot analysis of RNA isolated from primary cultures of normal human epidermal keratinocytes showed approximately 100-fold increased levels of the CYP1B1 mRNA after treatment with 10 nM TCDD for 24 h. Low levels of constitutive CYP1B1 mRNA were detected in 15 different human tissue samples. These results indicate that CYP1B1 is expressed in many normal human tissues and advance our understanding of the complexity of a gene family of cytochromes P450 whose expression is altered by TCDD.

The purpose of the present experiments was to examine dose-response relationships for induction of hepatic mRNA following a single administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats. The induction of cytochrome P450-1A1 (CYP1A1) mRNA is compared to other "dioxin-responsive" genes including UDP-glucuronosyltransferase I, plasminogen activator inhibitor 2, and transforming growth factor alpha using a sensitive reverse transcriptase-polymerase chain reaction-based method. Sample-to-sample variability in amplification is a concern in using polymerase chain reaction to quantitate biological responses. However, in the present study recombinant RNA templates were synthesized to use as internal standards in both the reverse transcription and the polymerase chain reaction steps. The induction of CYP1A1 mRNA was extremely sensitive to TCDD treatment with increases observed at doses as low as 1 ng/kg body weight. The induction of CYP1A1 mRNA correlated highly (R2 > 0.90) with an increase in ethoxyresorufin-o-deethylase activity, a CYP1A1-associated enzyme activity. However, induction of CYP1A1 mRNA levels was detected at lower TCDD doses than was ethoxyresorufin-o-deethylase activity, reflecting the greater sensitivity of the reverse transcription-polymerase chain reaction approach to detect transcriptional activation of the CYP1A1 gene. UDP-glucuronosyltransferase I mRNA was increased over control (5-fold) but required 1000-times higher TCDD doses (1 microgram/kg) to result in a significant increase than did CYP1A1. Plasminogen activator inhibitor 2 and transforming growth factor alpha mRNA, both previously shown to be induced by TCDD in human keratinocytes, were not increased in rat liver. Hence, these studies reaffirm that TCDD acts through classical receptor mechanisms with gene-to-gene differences in responsiveness. The reverse transcription-polymerase chain reaction method developed to measure mRNA for dioxin-responsive genes in rat liver will allow for measuring multigene and tissue responses to TCDD and other xenobiotics with high sensitivity, reproducibility, and adaptability and should increase our understanding of various dose-response relationships.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD), a widespread polychlorinated aromatic hydrocarbon, caused tumors in the liver and other sites when administered chronically to rats at doses as low as 0.01 microgram/kg/day. It functions in combination with a cellular protein, the Ah receptor, to alter gene regulation, and this resulting modulation of gene expression is believed to be obligatory for both dioxin toxicity and carcinogenicity. The U.S. EPA is reevaluating its dioxin risk assessment and, as part of this process, will be developing risk assessment approaches for chemicals, such as dioxin, whose toxicity is receptor-mediated. This paper describes a receptor-mediated physiologically based pharmacokinetic (PB-PK) model for the tissue distribution and enzyme-inducing properties of dioxin and discusses the potential role of these models in a biologically motivated risk assessment. In this model, ternary interactions among the Ah receptor, dioxin, and DNA binding sites lead to enhanced production of specific hepatic proteins. The model was used to examine the tissue disposition of dioxin and the induction of both a dioxin-binding protein (presumably, cytochrome P4501A2), and cytochrome P4501A1. Tumor promotion correlated more closely with predicted induction of P4501A1 than with induction of hepatic binding proteins. Although increased induction of these proteins is not expected to be causally related to tumor formation, these physiological dosimetry and gene-induction response models will be important for biologically motivated dioxin risk assessments in determining both target tissue dose of dioxin and gene products and in examining the relationship between these gene products and the cellular events more directly involved in tumor promotion.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent modulator of epithelial cell growth and differentiation, has been shown to induce transforming growth factor (TGF)-alpha in cultures of human keratinocytes and in the human keratinocyte cell line, SCC-12F. In this report, we investigated the mechanisms by which TCDD alters TGF-alpha expression. In addition, we studied the actions of TCDD on TGF-beta 1 and TGF-beta 2 expression. Treatment of SCC-12F cells with TCDD resulted in an increase in TGF-alpha and a reduction in TGF-beta 2 mRNA levels while mRNA levels for TGF-beta 1 were unchanged. Changes in TGF-alpha and TGF-beta 2 expression were maximal by 24 h. No change in the rate of transcription of TGF-alpha was detected following treatment with TCDD as determined by nuclear run-off analysis. TCDD treatment resulted in a stabilization of TGF-alpha mRNA as judged by an approximately 2-fold higher level of TGF-alpha mRNA in treated versus control cells in the presence of actinomycin D. In contrast to TGF-alpha, the rate of transcription of TGF-beta 2 was significantly reduced following TCDD treatment. These findings demonstrate that the induction of TGF-alpha expression in SCC-12F cells by TCDD occurs post-transcriptionally, primarily by mRNA stabilization, while TGF-beta 2 expression is reduced due to a decrease in the rate of TGF-beta 2 gene transcription.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), a widespread environmental contaminant, may elicit its effects by altering gene expression in susceptible cells. Five TCDD-responsive complementary DNA clones were isolated from a human keratinocyte cell line. One of these clones encodes plasminogen activator inhibitor-2, a factor that influences growth and differentiation by regulating proteolysis of the extracellular matrix. Another encodes the cytokine interleukin-1 beta. Thus, TCDD alters the expression of growth regulatory genes and has effects similar to those of other tumor-promoting agents that affect both inflammation and differentiation.

The phospholipase C (PLC)-mediated hydrolysis of membrane phosphoinositides is an important signal transduction pathway coupled to the cell-surface receptors for several hormones and growth factors. In addition, PLC activity can be modulated by changes in intracellular calcium and activation of GTP binding proteins. In this report, differential activation of PLC in the human keratinocyte cell line SCC-12F was studied as judged by specific patterns of inositol phosphate formation. Several hormones and growth factors previously shown to stimulate PLC in a variety of cell types were screened for activity in SCC-12F cells. Only bradykinin was active, stimulating the PLC-dependent generation of inositol (1,4,5) triphosphate (Ins(1,4,5)P3). Ins(1,4,5)P3 was rapidly metabolized to inositol(1,4)biphosphate (Ins(1,4)P2) and inositol(1,3,4,5)tetrakisphosphate (Ins(1,3,4,5)P4), and subsequently degraded to inositol monophosphates. The response elicited by bradykinin was concentration dependent (EC50 value of 50 nM), suggesting involvement of a specific bradykinin receptor. Treatment of these cells with the calcium ionophore A23187 appeared to result in the direct formation of Ins(1,4)P2 without Ins(1,4,5)P3 as precursor. Treatment of the cells with AIF4-, a putative activator of GTP binding proteins, resulted in the generation of inositol monophosphates as the major metabolites in the absence of detectable Ins(1,4,5)P3 formation. Taken together, these observations suggest that the PLC complex present in SCC-12F cells can be differentially activated to yield either Ins(1,4,5)P3, Ins(1,4)P2, or InsP. The observed effects may be due to a direct PLC-dependent hydrolysis of the appropriate membrane phosphoinositide.

A rapid and sensitive filtration assay for quantitating the specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to whole cells in culture is described. Cell monolayers are incubated with [3H]TCDD in the presence or absence of excess unlabeled ligand, detached from the culture dish with trypsin, filtered, and washed with cold (-78 degrees C) acetone to separate free and nonspecifically bound TCDD from specifically bound TCDD. TCDD receptor binding parameters were characterized in the murine hepatoma cell line Hepa1c1c7. The lower limit of detection of TCDD specific binding was in a sample equivalent to 10 micrograms of total cell protein. The equilibrium dissociation constant and stereospecificity for binding to the TCDD receptor were the same as those previously reported with other TCDD receptor assays on broken cell preparations. Analysis of binding in the murine hepatoma TCDD receptor variants TAO-c1BPrc1 and BPrc1 indicated that this assay will detect receptor number or affinity variants, but will not detect nuclear transfer deficient variants. The major advantage of the whole cell binding assay is that it provides the means to rapidly and reproducibly quantitate TCDD specific binding in small samples of whole cells in culture. In addition, this method eliminates loss or degradation of the receptor protein during the fractionation of cells required in previously reported methods. This method should prove useful in screening clonal cell populations for TCDD receptor number and affinity variants, and in screening for TCDD receptor binding activity in complementation studies of receptor deficient cells.

Continuous treatment of C3H/10T1/2 cells with low concentrations (greater than or equal to 4 pM) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhanced focus production in cultures pretreated with N-methyl-N'-nitro-N-nitrosoguanidine. Maximal enhancement occurred at 40 pM TCDD, a concentration 10 000-fold lower than that required to produce an optimal response with 12-O-tetradecanoylphorbol-13-acetate. Single treatments with 0.06 nM-5 microM TCDD did not transform C3H/10T1/2 cells or initiate the process of transformation in cultures subsequently exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Promotion of transformation is thus the predominant effect of TCDD in the C3H/101/2 cell transformation system.

The distribution of radioactivity associated with three 14C-labelled benzene metabolites was studied using whole body autoradiography (WBAR). Male Fischer-344 rats were given an intravenous dose of 0.6 mg/kg (60 microCi phenol, 1.2 mg/kg (100 microCi) catechol, or 1.3 mg/kg (100 microCi) hydroquinone. The rats were killed after 2 h and autoradiograms were prepared from whole body sagittal sections. The relative organ uptake of radioactivity associated with each compound was assessed by comparing tissue/blood optical density (O.D.) ratios from X-ray films. Bone marrow, thymus and the white pulp of the spleen concentrated radioactivity associated with hydroquinone or catechol. Radioactivity associated with phenol concentrated in the red pulp of the spleen, but not in the other lymphoid tissues. Radioactivity associated with all three metabolites was found in the lungs, kidneys and small intestines, whereas greater accumulation of radioactivity was observed in subcutaneous tissues, sebaceous glands and the white matter of the brain and spinal cord in rats given hydroquinone or catechol than in animals given phenol. Rats pretreated with a single dose of Aroclor 1254 (250 mg/kg, i.p.), a regimen which was found to protect against benzene-induced lymphocytopenia, were given hydroquinone (100 microCi; 1.3 mg/kg) or catechol (100 microCi; 1.4 mg/kg). For hydroquinone the tissue/blood O.D. ratios for bone marrow and thymus were approx. 60% lower in Aroclor-pretreated than in untreated rats. A 25% reduction in the tissue/blood O.D. ratios for these organs was observed in pretreated rats given catechol. These findings indicate that the uptake and concentration of radioactivity associated with hydroquinone and catechol by bone marrow and lymphoid organs (1) can occur independently of the metabolism of benzene in these tissues and (2) is reduced under conditions in which the animal is less susceptible to benzene toxicity.

Repeated administration of benzene (440 mg/kg/day, s.c.) to 6-week-old male Fischer-344 rats resulted in a progressive decline in the number of circulating lymphocytes. Pretreatment of these animals with 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) or 3,4,3',4'-tetrachlorobiphenyl (TCB) protected against benzene toxicity for as long as 7 days, but not after 10 days of repeated dosing. Representative phase I (mixed-function oxidase) and phase II (conjugating) enzyme activities were measured to determine whether the altered susceptibility to benzene toxicity in TCB- and HCB-pretreated rats could be correlated with changes in the profile of hepatic oxidative and detoxification pathways. Measurement of 7-ethoxycoumarin O-deethylase and benzphetamine N-demethylase activities indicated that the loss of protection by HCB or TCB against benzene toxicity after 7 days was not associated with changes in the activities of hepatic mixed-function oxidases inducible by 3-methylcholanthrene or phenobarbital. The time course for the stimulation by TCB and return to control values, of UDP-glucuronosyl transferase activity, a potential route for the elimination of benzene metabolites, mirrored the time course for the protection against toxicity. Epoxide hydratase activity was induced 2- to 3-fold by HCB. Although stimulation of this pathway could result in a decreased concentration of phenol, this activity did not decline with the loss of protection. Hepatic 10 000 X g supernatant fractions, prepared from livers of rats given TCB, were incubated with a non-saturating concentration of [14C] benzene (equivalent to 19 nmol/mg wet wt. tissue). Under these conditions the metabolism of benzene was depressed (40% of control) 2 days after pretreatment; after 14 days, the metabolism of benzene returned to control values. This pattern correlated temporarily with the protection against lymphocytopenia. The data indicate that the protection against benzene toxicity in rats pretreated with HCB or TCB is not necessarily related to the capacity of these compounds to induce phase I activities. In rats pretreated with TCB, the data suggest that decreasing the concentration of primary benzene metabolites, either by inhibiting the hepatic metabolism of benzene or increasing hepatic conjugation activity is an important factor modulating toxicity.

Blood cells are derived from a multitiered system of progenitor stem cells that lose their capacity for proliferation and self-renewal as they continue along pathways of differentiation. Since these hematopoietic events can be readily monitored in vivo and in vitro in the mouse, we have utilized this system to examine altered cellular differentiation associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Progenitor cells were suppressed following acute exposure of mice to TCDD at doses as low as 1.0 micrograms/kg body wt. In vitro studies demonstrated that myelotoxicity occurs by a direct inhibition of proliferating stem cells. Genetic studies indicated that the myelotoxic responses to TCDD, both in vivo and in vitro, segregate with the Ah locus. In addition, the in vitro myelotoxicity of various polyhalogenated aromatic hydrocarbon congeners correlated with their previously reported ability to induce hepatic microsomal enzyme activity and to bind to an intracellular receptor for TCDD. TCDD was also found to bind specifically to bone marrow cells from Ah-responsive, but not nonresponsive mice, indicating that bone marrow cells possess a specific receptor for TCDD. These data indicate that the myelotoxic response to TCDD is regulated by the Ah receptor present in the target tissue and demonstrates the utility of this system for examining the cellular and molecular events associated with the toxicity of polyhalogenated aromatic hydrocarbons, the prototype for which is TCDD.

An improved in vitro fluorometric assay of the O-deethylation of 7-ethoxycoumarin which is at least 10 times more sensitive than existing methods is described. Nearly quantitative recovery of the product which is essentially free of fluorescent impurities is obtained by a simple two-step extraction procedure. In C57BL/6J mice O-deethylating activity was induced 3-fold by 3-methylcholanthrene and 7-fold by phenobarbital. Administration of phenobarbital resulted in a 10-fold stimulation of enzyme activity in DBA/2J mice. Kinetic analysis indicated that at least two and possibly more components are involved in the metabolism of 7-ethoxycoumarin. Differential stimulation of the maximal activities associated with these components was observed after the administration of phenobarbital, 3-methylcholanthrene, or 2,3,7,8-tetrachlorodibenzo-p-dioxin. The ED50 values for the induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities in C57BL/6J mice were almost identical and approximately 10-fold less than in DBA/2J mice similarly treated.

The responsiveness of 5 human squamous cell carcinoma (SCC) lines derived from tumors of the epidermis and tongue to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was assessed by measuring the induction of the cytochrome P1-450-mediated monooxygenase activity, 7-ethoxycoumarin O-deethylase (ECOD). In 4 of the SCC lines the EC50 for this response was approximately 10(-9)M, whereas in one line the EC50 was 10(-10)M. In each of the less sensitive lines a concentration of 10(-10)M TCDD elicited less than 5% of the maximal enzyme activity. Specific binding of radiolabeled TCDD was detected in the cytosol fraction from all the SCC lines. The relative amount of receptor measured in each line correlated with maximally-induced ECOD activity. The data indicate that human cell lines derived from a target tissue for TCDD toxicity contain the TCDD receptor and show differential sensitivity to TCDD analogous to the murine strain differences in sensitivity regulated by the Ah locus.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) modulates the proliferation, differentiation, or both, of epidermal keratinocytes in vivo and in culture. The growth of epidermal cells in culture is regulated by several biochemical mediators including epidermal growth factor (EGF). In this report the actions of TCDD on EGF binding in a basal cell population from a human keratinocyte cell line were examined. TCDD decreased the specific binding of 125I-EGF to basal cells by 40% within 96 hr. This reduction in EGF binding could not be attributed to changes in the state of differentiation as assessed by cell size and morphology, and cornified envelope competence, a marker of terminal differentiation. Modulation of EGF binding by TCDD was concentration-dependent (EC50 = 1 nM) and stereospecific, suggesting involvement of the Ah receptor. Scatchard analysis of EGF binding to the basal cells indicated a single class of high-affinity sites in both control (Kd = 0.14 nM) and treated (Kd = 0.11 nM) cultures and confirmed a decrease in the number of these sites in response to TCDD. The reduction in EGF binding correlated with a decrease in EGF-stimulated DNA synthesis and cell proliferation. Comparison of differentiation-competent squamous cell carcinoma (SCC) lines treated with TCDD supported an association between modulation of EGF binding and enhanced differentiation. The data indicate that basal cells are a target for TCDD. We propose that the modulation of EGF binding in basal keratinocytes by TCDD is one of the critical regulatory events resulting in enhanced differentiation.

Specific toxic and biochemical responses elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human thymic epithelial (TE) cells in culture are mediated by the TCDD receptor protein. Characterization of the physicochemical properties of the TCDD receptor in cytosol fractions from cultured human TE cells indicates that this protein is similar, but not identical, to the extensively studied receptor species present in mouse and rat liver. The human TCDD receptor sediments at 9.1 S on sucrose density gradients at 0 degrees, undergoes a temperature-dependent conversion at 20 degrees to a species sedimenting at 10.7 S, and partially dissociates in the presence of 0.4 M KCl, as judged by the appearance of a peak sedimenting at 5 S. Both the temperature- and salt-dependent changes in the observed physical properties of the human TCDD receptor are inhibited by sodium molybdate. Two molybdate-stabilized binding species can be resolved from TCDD specific binding isotherms to human TE cytosol. Under identical conditions, only a single TCDD specific binding component was detected in cytosol fractions from both mouse and rat liver. Mixing cytosol prepared from human TE cells with hepatic cytosol fractions from C57BL/6 mice revealed the presence of a heat-labile, trypsin-sensitive factor in human TE cells that inhibits TCDD specific binding to the murine hepatic receptor. Molybdate stabilized the mouse receptor against the actions of this human inhibitory factor. Molybdate also may stabilize the human TCDD receptor, as indicated by the 2- to 3-fold increase in total TCDD specific binding measured in human TE cytosol fractions in the presence of this metallo-oxyanion.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and isosteric halogenated analogs produce a spectrum of pathologic changes in the epidermis of humans. In this study, the actions of TCDD on cultured human epidermal cells were characterized to determine whether these cells are an appropriate in vitro model to examine the mechanisms of TCDD toxicity to human skin. The differential staining properties of TCDD-treated cultures indicated that TCDD decreased basal cell numbers and increased the degree of keratinization. Histologic examination of cross-sections of the cultures confirmed a loss of small nucleated cells and increased cell layering in response to TCDD. TCDD produced no change in total cell number or cell protein, but decreased the number of small (basal) cells and DNA synthesis. TCDD increased the number of cells containing spontaneous envelopes, as well as the number of envelope-competent cells. The quantitative changes observed in these parameters were consistent with a TCDD-induced commitment of proliferating cells to terminal differentiation. TCDD also decreased epidermal growth factor (EGF) specific binding. Maximal changes in EGF binding occurred after 4 days, and in small cell number after 5 days. The decreases in EGF binding and small cell number were stereospecific and concentration dependent (EC50, 1 to 2 nM), implicating the human Ah receptor in mediating these responses to TCDD. These data indicate that TCDD treatment produces hyperkeratinization in cultured human epidermal cells. It is proposed that TCDD acts on epidermal basal cells to enhance terminal differentiation through mechanisms regulated at least in part by the Ah receptor.

Studies of the toxic actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in numerous animal models and in human and animal cells in culture have established that the most characteristic pathologic lesions produced by this compound result from events initiated by the interaction of TCDD with a specific intracellular receptor protein, the Ah receptor. Although most research on the interaction of TCDD with the Ah receptor has focused on establishing involvement of this receptor complex in specific toxic responses, recent application of modern cell and molecular biology techniques is yielding new insights into the mechanism(s) of signal transduction. Elucidation of these mechanisms is essential for understanding the molecular basis of the cell and species specificity which is a hallmark of TCDD toxicity. This knowledge should provide the framework for development of a more toxicologically relevant risk assessment model.

It has been proposed [J. D. McKinney, J. Fawkes, S. Jordan, K. Chae, S. Oatley, R. E. Coleman, and W. Briner (1985). Environ. Health Perspect. 61, 41-53] that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces toxic responses through persistent occupancy of nuclear thyroxine (T4) receptors, and that maintenance of receptor occupancy by supraphysiologic concentrations of thyroid hormones mimics TCDD toxicity [L. H. Hong, J. D. McKinney, and M. I. Luster (1987). Biochem. Pharmacol., 36, 1361-1365]. TCDD induces hyperkeratinization in cultured normal human epidermal cells and the human keratinocyte line, SCC-12F. This response is associated with a decrease in high-affinity epidermal growth factor (EGF) receptors. These cell systems were used as models to compare the actions of TCDD with those of triiodothyronine (T3) and T4 on human target cells. Keratinocytes were treated simultaneously with T3 and T4 in a 4:1 molar ratio (T3/T4; Hong et al., 1987) and levels of EGF binding and 7-ethoxycoumarin O-deethylase activity (a marker for cytochrome P1-450 induction) were measured. T3/T4 (at concentrations up to 10 microM T3/2.5 microM T4) and T3 or T4 alone (0.1 to 10 microM) did not produce the hyperkeratinization, the decrease in EGF binding, or the increase in ECOD activity that are characteristic of TCDD exposure. Nonresponsiveness to T3/T4 was not due to metabolism of these hormones by the keratinocytes. T3 and T4 did not compete with [3H]TCDD for binding to cytosolic Ah receptor from C57BL6 mouse liver, SCC-12F, or normal human epidermal cells. TCDD and an active stereoisomer, 2,3,7,8-tetrachlorodibenzofuran, did not compete with [125I]T3 or [125I]T4 for binding to nuclear receptors from SCC-12F cells or C57BL6 mouse liver. Taken together, these data demonstrate that the actions of TCDD and thyroid hormones are mediated by distinct mechanisms in human keratinocytes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) acts on selected targets within the immune system to produce a characteristic profile of pathologic responses typified by thymic atrophy, suppressed cellular immunity, and inhibition of antibody production to T-lymphocyte-dependent antigens. Studies in inbred mice differing in sensitivity to TCDD indicate that TCDD-induced thymic atrophy is mediated by a receptor protein (designated the Ah receptor). To study the cellular and molecular basis for TCDD-induced thymic atrophy, primary cultures of thymic epithelial (TE) cells were established from C57BL/6 mice, a strain sensitive to TCDD. Treatment of TE monolayers with TCDD (0.1 to 10 nM) resulted in the altered maturation of cocultured syngeneic thymocytes as judged by suppression (40% of control at 10 nM TCDD) of TE-dependent responsiveness of thymocytes to the mitogens concanavalin A and phytohemagglutinin. TE-conditioned medium enhanced the mitogen responsiveness of thymocytes three- to four-fold; however, the enhanced mitogen response mediated by the TE-conditioned medium was not suppressed in thymocytes incubated in medium collected from TCDD-treated cultures or in TE-conditioned medium to which TCDD (10 nM) had been added directly. The suppression of TE-dependent maturation of thymocytes was concentration dependent (EC50 approximately 1 nM) and stereospecific, suggesting involvement of the Ah receptor. The Ah receptor in cytosol fractions from cultured TE cells was measured directly and was found to be present at a concentration 3 and 3.5 times greater than that measured in whole thymus and thymocytes, respectively. The results of this study indicate that TCDD can act directly on epithelial target cells in the thymus: one consequence of this action appears to be the altered thymus-dependent maturation of T-lymphocyte precursors, mediated through direct cell-cell contact between thymocytes and TE cells.

A coculture system of human thymic epithelial (HuTE) cells and thymocytes (T lymphocyte precursors) has been established and characterized as an in vitro model for assessing the potential toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to human thymus. HuTE cells in culture were responsive to TCDD as judged by induction of the cytochrome P1-450 monooxygenase activities, 7-ethoxycoumarin O-deethylase (ECOD) and 7-ethoxyresorufin O-deethylase (EROD). Measurement of the responsiveness of thymocytes cocultured on TCDD-pretreated HuTE monolayers to the mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) indicated that TCDD can act directly on HuTE cells to suppress thymocyte maturation (at a concentration of 10 nM, TCDD produced a 25 to 50% inhibition of thymocyte responsiveness to Con A and PHA). Both the induction of cytochrome P1-450 monooxygenase activity (EC50 values approximately 1 nM) and immunosuppressive responses elicited by TCDD in HuTE cells were concentration-dependent and stereospecific (as judged by the relative activities of chlorinated dibenzo-p-dioxin and dibenzofuran isomers), indicating involvement of the Ah receptor which was detected in all HuTE strains examined. Initial characterization of these Ah receptor-mediated responses in several strains of HuTE cells indicated marked interstrain differences in maximally inducible ECOD and EROD activities which did not appear to directly correlate with measured concentrations of the cytosolic Ah receptor, and in certain strains examined, differences in sensitivity and magnitude were observed for TCDD-evoked immunotoxic responses but not always for the induction response. These data on the actions of TCDD on cultured HuTE cells suggest that human thymus is a target for TCDD and related halogenated aromatic compounds. In HuTE cells, measurement of either the Ah receptor concentration or of marker responses such as the induction of cytochrome P1-450 alone cannot provide an accurate quantitative assessment of susceptibility to TCDD-induced thymus toxicity.

Human exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and chlorinated analogs commonly results in pathological changes in the skin and its appendages characterized by thickening of the epidermis (acanthosis), hyperkeratosis and squamous metaplasia of the epithelial lining of the sebaceous glands. Acneform lesions (chloracne) develop as hair follicles dilate and fill with keratin and sebaceous glands become cystic. In animal models it has been found that the chloracneogenic potential of the halogenated aromatic compounds examined corresponds with the relative affinity of these same compounds for the cytosolic TCDD receptor. This receptor controls the coordinate expression of a number of inducible enzyme activities and in certain cell targets can alter normal programs of proliferation and differentiation. In this report we describe some of our ongoing studies on the mechanisms of action of TCDD in normal human epidermal cells and squamous cell carcinoma (SCC) lines. These systems permit detailed investigation of the molecular and biochemical events underlying pathologic changes in the skin and offer the potential of establishing a risk assessment model for halogenated aromatic compounds by using human target cells.

It was recently reported that suppression of murine bone marrow hematopoiesis is a very sensitive indicator for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity (1). We report here that a structural analog of TCDD, 1-NH2-3,7,8-trichlorodibenzo-p-dioxin (NH2-TriCDD), is a specific and effective antagonist for TCDD-induced myelotoxicity and enzyme induction. When administered to mice or added directly into culture at a 100-fold excess, relative to TCDD, NH2-TriCDD completely abrogated the ability of TCDD to inhibit granulocyte-macrophage progenitor cells (CFU-C) formation, an indicator of hematopoiesis. Further, NH2-TriCDD inhibited TCDD-induced activation of cytochrome P1-450 monooxygenase activity. Studies designed to measure specific binding of TCDD to the cytosolic Ah receptor indicated that NH2-TriCDD effectively inhibited binding of TCDD to the receptor by acting as a competitive antagonist (Ki = 0.72 nM).