Aromatic Hydrocarbon Receptor Agonist Research Articles

Constitutive expression of AhR and BRCA-1 promoter CpG hypermethylation as biomarkers of ERα-negative breast tumorigenesis.

BackgroundOnly 5–10 % of breast cancer cases is linked to germline mutations in the BRCA-1 gene and occurs early in life. Conversely, sporadic breast tumors, which represent 90-95 % of breast malignancies, have lower BRCA-1 expression, but not mutated BRCA-1 gene, and tend to occur later in life in combination with other genetic alterations and/or environmental exposures. The latter may include environmental and dietary factors that activate the aromatic hydrocarbon receptor (AhR). Therefore, understanding if changes in expression and/or activation of the AhR are associated with somatic inactivation of the BRCA-1 gene may provide clues for breast cancer therapy.MethodsWe evaluated Brca-1 CpG promoter methylation and expression in mammary tumors induced in Sprague–Dawley rats with the AhR agonist and mammary carcinogen 7,12-dimethyl-benzo(a)anthracene (DMBA). Also, we tested in human estrogen receptor (ER)α-negative sporadic UACC-3199 and ERα-positive MCF-7 breast cancer cells carrying respectively, hyper- and hypomethylated BRCA-1 gene, if the treatment with the AhR antagonist α-naphthoflavone (αNF) modulated BRCA-1 and ERα expression. Finally, we examined the association between expression of AhR and BRCA-1 promoter CpG methylation in human triple-negative (TNBC), luminal-A (LUM-A), LUM-B, and epidermal growth factor receptor-2 (HER-2)-positive breast tumor samples.ResultsMammary tumors induced with DMBA had reduced BRCA-1 and ERα expression; higher Brca-1 promoter CpG methylation; increased expression of Ahr and its downstream target Cyp1b1; and higher proliferation markers Ccnd1 (cyclin D1) and Cdk4. In human UACC-3199 cells, low BRCA-1 was paralleled by constitutive high AhR expression; the treatment with αNF rescued BRCA-1 and ERα, while enhancing preferential expression of CYP1A1 compared to CYP1B1. Conversely, in MCF-7 cells, αNF antagonized estradiol-dependent activation of BRCA-1 without effects on expression of ERα. TNBC exhibited increased basal AhR and BRCA-1 promoter CpG methylation compared to LUM-A, LUM-B, and HER-2-positive breast tumors.ConclusionsConstitutive AhR expression coupled to BRCA-1 promoter CpG hypermethylation may be predictive markers of ERα-negative breast tumor development. Regimens based on selected AhR modulators (SAhRMs) may be useful for therapy against ERα-negative tumors, and possibly, TNBC with increased AhR and hypermethylated BRCA-1 gene.

Gestational exposure to the AhR agonist 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin induces BRCA‐1 promoter hypermethylation and reduces BRCA‐1 expression in mammary tissue of rat offspring: Preventive effects of resveratrol

Studies with murine models suggest that maternal exposure to aromatic hydrocarbon receptor (AhR) agonists may impair mammary gland differentiation and increase the susceptibility to mammary carcinogenesis in offspring. However, the molecular mechanisms responsible for these perturbations remain largely unknown. Previously, we reported that the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CpG methylation of the breast cancer-1 (BRCA-1) gene and reduced BRCA-1 expression in breast cancer cell lines. Based on the information both the human and rat BRCA-1 genes harbor xenobiotic responsive elements (XRE = 5'-GCGTG-3'), which are binding targets for the AhR, we extended our studies to the analysis of offspring of pregnant Sprague-Dawley rats treated during gestation with TCDD alone or in combination with the dietary AhR antagonist resveratrol (Res). We report that the in utero exposure to TCDD increased the number of terminal end buds (TEB) and reduced BRCA-1 expression in mammary tissue of offspring. The treatment with TCDD induced occupancy of the BRCA-1 promoter by DNA methyltransferase-1 (DNMT-1), CpG methylation of the BRCA-1 promoter, and expression of cyclin D1 and cyclin-dependent kinase-4 (CDK4). These changes were partially overridden by pre-exposure to Res, which stimulated the expression of the AhR repressor (AhRR) and its recruitment to the BRCA-1 gene. These findings point to maternal exposure to AhR agonists as a risk factor for breast cancer in offspring through epigenetic inhibition of BRCA-1 expression, whereas dietary antagonists of the AhR may exert protective effects.

BRCA-1 promoter hypermethylation and silencing induced by the aromatic hydrocarbon receptor-ligand TCDD are prevented by resveratrol in MCF-7 Cells

Epigenetic mechanisms may contribute to reduced expression of the tumor suppressor gene BRCA-1 in sporadic breast cancers. Through environmental exposure and diet, humans are exposed to xenobiotics and food compounds that bind the aromatic hydrocarbon receptor (AhR). AhR-ligands include the dioxin-like and tumor promoter 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XREs=GCGTG) and interactions with transcription cofactors. Previously, we reported on the presence of several XREs in the proximal BRCA-1 promoter and that the expression of endogenous AhR was required for silencing of BRCA-1 expression by TCDD. Here, we document that in estrogen receptor-α-positive and BRCA-1 wild-type MCF-7 breast cancer cells, the treatment with TCDD attenuated 17β-estradiol-dependent stimulation of BRCA-1 protein and induced hypermethylation of a CpG island spanning the BRCA-1 transcriptional start site of exon-1a. Additionally, we found that TCDD enhanced the association of the AhR; DNA methyl transferase (DNMT)1, DNMT3a and DNMT3b; methyl binding protein (MBD)2; and trimethylated H3K9 (H3K9me3) with the BRCA-1 promoter. Conversely, the phytoalexin resveratrol, selected as a prototype dietary AhR antagonist, antagonized at physiologically relevant doses (1 μmol/L) the TCDD-induced repression of BRCA-1 protein, BRCA-1 promoter methylation and the recruitment of the AhR, MBD2, H3K9me3 and DNMTs (1, 3a and 3b). Taken together, these observations provide mechanistic evidence for AhR agonists in the establishment of BRCA-1 promoter hypermethylation and the basis for the development of prevention strategies based on AhR antagonists.

Abstract 5419: Effects of selective aryl hydrocarbon receptor modulators (SAhRMs) in enhancing activity of tamoxifen against doxorubicin resistant MCF-7 Cells: Role of BRCA1

Abstract Recent reports indicate that SAhRMs such as diindolylmethane (DIM) inhibit α-estrogen receptor (ER) expression and estrogen signaling in human breast cancer cells. There is also increasing evidence that SAhRMs demonstrate anti estrogenic and antitumorigenic activity in ER positive human breast adenocarcinoma (MCF-7) cells via cross talk between aromatic hydrocarbon receptor (AhR) and ER pathways. Additional reports suggest that BRCA1 transcriptional activity is enhanced by interactions with AhR and the levels of BRCA1 expression play a role in tamoxifen sensitivity/resistance in breast cancer cells by altering estrogen receptor-coregulator interactions. However, the effects of AhR and its agonists and antagonists, and their modulatory effect on BRCA1 in resistant human breast cancer cells have not been investigated. We have tested the hypothesis that DIM, an AhR agonist may inhibit cell growth in a concentration and time dependant manner against doxorubicin resistant MCF-7 (MCF-7/dox) cells and may enhance the activity of 4-OH tamoxifen (TAM) against both MCF-7 and MCF-7/dox cells by enhancing the expression of BRCA1. Initially we determined the cytotoxic effect of DIM against the two cell lines by utilizing the MTT assay. Both DIM and TAM were equally effective against MCF-7 and MCF-7/dox cells with the IC50 values of approximately 90 μM and 20 μM, respectively at 72 hr. However, a combination of DIM and TAM resulted in an increase in cytotoxcity and statistically significant lowering of the IC50 concentration to 50 μM and 10 μM for each drug, respectively. We also investigated the effects of AhR antagonist, α-naphthoflavone (NF) which had a similar IC 50 value of approximately 112 μM against both these cell lines, whereas a combination of NF and TAM required only half the concentrations of 50 μM and 10 μM to inhibit 50 % of the cell growth of each cell line. Since BRCA1, a tumor suppressor gene, has been implicated in the mechanism of tamoxifen induced growth inhibition, we determined the expression of this protein upon exposure of these cells to DIM. Results from western blot analysis show that there is a significant increase in BRCA1 and AhR protein levels in these cells upon treatment with DIM when compared to protein levels from untreated MCF-7 and MCF-7 /dox cells. Thus activation of BRCA1 by SAhRMs appears to be one of the mechanisms responsible for enhanced growth inhibitory effect of tamoxifen against these cells. These data suggest that either an AhR agonist or an antagonist can be conveniently employed in combination with TAM to increase the activity of TAM against both MCF-7 cells and doxorubicin resistant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5419.

In vivo and in vitro Ah-receptor activation by commercial and fractionated pentabromodiphenylether using zebrafish ( Danio rerio) and the DR-CALUX assay

The present study addresses the toxicity of a commercial pentabrominated diphenylether (PeBDE) flame retardant mixture, DE-71, in a model aquatic vertebrate. Four weeks’ exposure of juvenile zebrafish ( Danio rerio) to water-borne DE-71 resulted in dose-dependent induction of CYP1A immunoreactivity, predominantly in the endocardium and the endothelium of larger blood vessels, such as ventral aorta and branchial arteries, as well as the larger hepatic and pancreatic blood vessels. To investigate the impact of possible contaminating PBDD/Fs in the DE-71 product, the study was repeated after DE-71 had been fractionated into a non-planar (cleaned PBDEs) and a planar fraction (PBDD/Fs). Zebrafish were exposed under similar conditions to the planar and cleaned DE-71 fractions, and to uncleaned DE-71. In addition, the above fractions were chemically analyzed and tested in a reporter gene assay (DR-CALUX) for their aromatic hydrocarbon-receptor (AhR) stimulating potencies. A relatively strong CALUX response was detected from the planar DE-71 fraction (19.7 ng TCDD equivalent (TEQ)/g DE-71), coinciding with a strong induction of CYP1A immunoreactivity in zebrafish. CYP1A immunoreactivity in zebrafish exposed to uncleaned DE-71 was intense, although the CALUX response was 10-fold less compared to the planar fraction. Only weak CYP1A immunoreactivity was found in fish exposed to cleaned DE-71, and none in control animals; no CALUX response was detected in cleaned DE-71. The present findings indicate that chemical impurities of the commercial PeBDE product account for AhR-mediated effects. Analytical isolation of a planar fraction from the commercial product increased the in vitro (DR-CALUX) signal 10 times. Immunohistochemistry showed a strong tissue specific reaction to DE-71 in vivo at these relatively low TEQ levels regardless of chemical pretreatment of the mix, reflecting the sensitivity of CYP1A induction in juvenile zebrafish to AhR agonists.

Induction of the transferrin receptor gene by benzo[a]pyrene in breast cancer MCF‐7 cells: Potential as a biomarker of PAH exposure

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental DNA-damaging agents regarded as risk factors for human disease, including lung and breast cancer. The biotransformation of PAHs to carcinogenic metabolites is mediated by the aromatic hydrocarbon receptor (AhR), which activates transcription at xenobiotic responsive elements (XREs = 5'-GCGTG-3') found in the promoter regions of genes encoding for detoxifying enzymes, including CYP1A1 and CYP1B1. In this study, we wished to identify novel biomarkers that may be useful in monitoring critical carcinogenic events of the breast induced by PAHs. Using a GeneMAP CancerArray, we analyzed in breast cancer MCF-7 cells the temporal effects of the AhR agonist benzo[a]pyrene (B[a]P), which is a prototype PAH and known environmental carcinogen. Genes upregulated at least threefold by B[a]P and containing potential XREs within their promoter regions included CYP1A1, CYP1B1, paired box gene 3 (PAX3), cortactin (CTTN/EMS1), beta-2-microglobulin (B2M), and transferrin receptor (TfR). The stimulatory effects of B[a]P on expression of these genes were abrogated by cotreatment with the AhR antagonist flavonoid, alpha-napthoflavone (ANF). The TfR gene was selected for further analysis as its promoter region contains two potential XREs and its expression has been shown to be increased in breast cancer cells. Accumulation of TfR mRNA in B[a]P-treated cells was confirmed by quantitative real time PCR. Transient transfection studies indicated that the transcriptional activity of the TfR promoter was stimulated by B[a]P, whereas ANF counteracted this induction. These results indicate that the TfR gene may be a potential biomarker of PAH exposure.

Aryl hydrocarbon receptor (AhR) agonists suppress interleukin-6 expression by bone marrow stromal cells: an immunotoxicology study.

BackgroundBone marrow stromal cells produce cytokines required for the normal growth and development of all eight hematopoietic cell lineages. Aberrant cytokine production by stromal cells contributes to blood cell dyscrasias. Consequently, factors that alter stromal cell cytokine production may significantly compromise the development of normal blood cells. We have shown that environmental chemicals, such as aromatic hydrocarbon receptor (AhR) agonists, suppress B lymphopoiesis by modulating bone marrow stromal cell function. Here, we extend these studies to evaluate the potential for two prototypic AhR agonists, 7,12-dimethylbenz [a]anthracene (DMBA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to alter stromal cell cytokine responses.MethodsBone marrow stromal cells were treated with AhR agonists and bacterial lipopolysaccharide (LPS) to mimic innate inflammatory cytokine responses and to study the effects of AhR ligands on those responses. Steady state cytokine RNA levels were screened by RNAse protection assays (RPA) and quantified by real-time PCR. Cytokine (IL-6) protein production was measured by ELISA. NF-κB EMSAs were used to study IL-6 transcriptional regulation.ResultsRPAs indicated that AhR+ bone marrow stromal cells consistently up-regulated genes encoding IL-6 and LIF in response to LPS, presumably through activation of Toll-like receptor 4. Pre-treatment with low doses of DMBA or TCDD selectively abrogated IL-6 gene induction but had no effect on LIF mRNA. Real-time-PCR indicated a significant inhibition of IL-6 mRNA by AhR ligands within 1 hour of LPS challenge which was reflected in a profound down-regulation of IL-6 protein induction, with DMBA and TCDD suppressing IL-6 levels as much as 65% and 88%, respectively. This potent inhibitory effect persisted for at least 72 hours. EMSAs measuring NF-κB binding to IL-6 promoter sequences, an event known to induce IL-6 transcription, indicated a significant decrease in the LPS-mediated induction of DNA-binding RelA/p50 and c-Rel/p50 heterodimers in the presence of DMBA.ConclusionsCommon environmental AhR agonists can suppress the response to bacterial lipopolysaccharide, a model for innate inflammatory responses, through down-regulation of IL-6, a cytokine critical to the growth of several hematopoietic cell subsets, including early B cells. This suppression occurs at least at the level of IL-6 gene transcription and may be regulated by NF-κB.

Induction and superinduction of 2,3,7,8-tetrachlorodibenzo- p-dioxin-inducible poly(ADP-ribose) polymerase: Role of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator transcription activation domains and a labile transcription repressor

The environmental contaminant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) induces a novel poly(ADP-ribose) polymerase (TiPARP). In this study, the signaling pathway of the induction was analyzed. Induction of TiPARP by TCDD occurs in both hepa1c1c7 cells and C57 mouse liver. Induction is concentration and time dependent. Genetic analyses reveal that induction is abolished in aromatic hydrocarbon receptor (AhR)- or aromatic hydrocarbon receptor nuclear translocator (Arnt)-defective variants but restored upon reconstitution of the variant cells with cDNAs expressing functional AhR or Arnt. Moreover, induction is largely reduced in cells expressing a deletion mutant of AhR or Arnt lacking the transcription activation (TA) domain, thus implicating the TA activities of both AhR and Arnt in the induction. Inhibition of protein synthesis by cycloheximide enhances the induction of TiPARP in the presence of an AhR agonist. The superinduction is transcriptional and does not require pretreatment with TCDD. Finally, inhibition of the 26S proteasomes by MG132 superinduces TiPARP. These findings establish that induction of TiPARP by TCDD is mediated through an AhR and Arnt transcription activation-dependent signal transduction that is repressed by a labile factor through the ubiquitin–26S proteasome-mediated protein degradation.

Lack of effect of beta-naphthoflavone on induction of Nramp genes in adult rainbow trout Oncorhynchus mykiss

Natural resistance-associated macrophage protein (Nramp) genes in rainbow trout, Oncorhynchus mykiss, were identified and characterized. The greatest mRNA level encoding these genes was in the developing ovary of rainbow trout. We evaluated the response of these genes to a certain aromatic hydrocarbon receptor (AHR) agonist. Adult rainbow trout were treated with β-naphthoflavone (BNF) (50 and 100 mg/kg) for 48 h. Using reverse-transcriptase polymerase chain reaction with ovary and head kidney RNA and specific α and β Nramp primers, a 400 bp Nramp-α- and a 400 bp Nramp-β-specific cDNA were obtained. There were no changes in the α and β Nramp mRNA levels in the ovary following BNF administration. CYP1A1 mRNA was increased in the ovary and kidney, suggesting the presence of AHR in rainbow trout ovary, while the AHR agonist produced no effect on Nramp mRNAs.

Ah Receptor-Based Chemical Screening Bioassays: Application and Limitations for the Detection of Ah Receptor Agonists

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biologic and toxicologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Here we utilized two AhR-dependent bioassay systems as screening tools to identify novel AhR agonists and to detect the presence of AhR agonists in sample extracts. These assays measure the ability of a chemical to activate AhR DNA binding in vitro (GRAB bioassay) or AhR-dependent (luciferase) gene expression in cultured cells (CALUX bioassay). Known AhR agonists (halogenated and nonhalogenated aromatic hydrocarbons) were positive in both assays, whereas the AhR antagonist alpha-naphthoflavone exhibited agonist activity only in the GRAB assay. In vitro GRAB analysis has identified several imidazoline receptor ligands and beta-carbolines as AhR agonists and also revealed the presence of AhR agonist activity in crude DMSO extracts of commercial newspapers. In contrast to their positive activity in the GRAB assay, the majority of these chemicals/extracts were only weakly active or inactive in the cell-based CALUX assay. Our results not only reveal that the ability of a chemical to activate the AhR in vitro does not necessarily correlate with its ability to induce gene expression in intact cells, but the high level of false positives obtained with the GRAB assay clearly demonstrates its inability to accurately identify AhR agonists or agonist activity. Screening of unknown chemicals, chemical classes, and samples for AhR agonist activity will require the use of intact cell bioassays.

Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells.

Human cytochromes P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) catalyze the metabolic activation of a number of procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. The aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a marked effect on estrogen metabolism in MCF-7 breast-tumor cells by induction of these two enzymes. To investigate whether induction of CYP1A1 and CYP1B1 by AhR agonists and the associated increase in E2 metabolism are common to all breast epithelial cells and breast-tumor cells, we determined the effects of TCDD on E2 metabolism, and CYP1A1 and CYP1B1 mRNA levels in a series of non-tumor-derived breast epithelial (184A1 and MCF-10A) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cell lines. In 184A1 cells, which did not express detectable estrogen receptor (ER) alpha mRNA, CYP1A1 mRNA and activity were induced by TCDD, and enhanced E2 metabolism in TCDD-treated cells was predominantly E2 2-hydroxylation. In MCF-10A, MCF-7, T-47D, ZR-75-1 and BT-20 cells, which expressed varying levels of ER alpha mRNA, both CYP1A1 and CYP1B1 mRNA levels and rates of both E2 2- and 4-hydroxylation were highly elevated following exposure to TCDD. In MDA-MB-157, MDA-MB-231 and MDA-MB-436 cells, which did not express detectable ER alpha mRNA and generally displayed fibroblastic or mesenchymal rather than epithelial morphology, CYP1B1 induction was favored, and the rate of E2 4-hydroxylation exceeded that of 2-hydroxylation in TCDD-treated cells. These results show that breast epithelial cells and tumor cells vary widely with regard to AhR-mediated CYP1A1 and CYP1B1 induction, suggesting that factors in addition to the AhR regulate CYP1A1 and CYP1B1 gene expression. In these cell lines, significant CYP1A1 inducibility was restricted to cultures displaying epithelial morphology, whereas CYP1B1 inducibility was observed in cells of both epithelial and mesenchymal morphology.

Genetic differences in alcohol drinking preference between inbred strains of mice.

Genetic factors are known to influence the preference for drinking alcohol-in humans as well as certain inbred strains of laboratory animals. Here we examined the possible role of the aromatic hydrocarbon receptor (AHR) in alcohol-preferring C57BL/6J (B6, high-affinity AHR) and alcohol-avoiding DBA/2J (D2, low-affinity AHR) inbred mouse strains, and in the two congenic lines B6.D2-Ahrd (> 99% B6 genome with the D2 low-affinity AHR) and D2.B6-Ahrb-1 (> 99% D2 genome with the B6 high-affinity AHR). This laboratory had previously shown an association between resistance to intraperitoneal ethanol-induced toxicity and the high-affinity AHR. Offering the choice between drinking water and 10% ethanol, we found that alcohol preference is three- to four-fold greater in B6 than D2 mice, as well as three- to four-fold greater in B6.D2-Ahrd than D2.B6-Ahrb-1 mice-indicating that alcohol preference is AHR-independent. The prototype AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) did not affect the rates of chronic alcohol consumption in B6 or D2 mice, suggesting that dioxin-inducible metabolism does not play a major role in alcohol drinking preference. In B6 mice, we found that oral treatment with the aldehyde dehydrogenase (ALDH) inhibitor disulfiram decreased alcohol preference by 50%, whereas oral treatment of the catalase inhibitor 3-amino-1,2,4-triazole increased alcohol drinking preference by 15-20%. Although liver and brain ALDH activities were both significantly higher in D2 than B6, these activities were not related to alcohol consumption. Hepatic and brain catalase activities, on the other hand, were two- to three-fold higher in D2 and D2.B6-Ahrb-1 mice, compared with that in B6 and B6.D2-Ahrd. Furthermore, brain acetaldehyde levels were inversely related to the quantity of alcohol voluntarily consumed. We conclude that the alcohol drinking preference between the B6 and D2 inbred mouse strains is independent of the Ah receptor-but is genetically determined, in part, by the level of brain catalase activity which, in turn, regulates brain acetaldehyde concentrations.

Regulation of the Aromatic Hydrocarbon Receptor (AHR) by In-Utero and Lactational Exposure to 2, 3, 7, 8-Tetrachlorodibenzo-p-Dioxin (TCDD).

The aromatic hydrocarbon receptor (AHR) mediates the toxicity of several halogenated aromatic hydrocarbons, including 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In order to better understand the systemic endocrine disruption induced by AHR agonists, we examined the regulation of AHR by exposure to TCDD during gestation and lactation. Pregnant rats were given an oral dose of 1 μg TCDD/kg body weight or vehicle control on day 15 of gestation, and female pups sacrificed on post-natal day 21. Total RNA from ovaries, uteri, and hypothalami was immobilized on a nylon membrane and probed with a murine AHR cDNA. The gel mobility-shift assay was used to asses changes in the DNA-binding activity of AHR in vitro. Ovarian AHR mRNA and DNA-binding capability were decreased 2.1- and 1.7-fold, respectively; while in the uterus, there was a 1.3- and 1.5-fold increase in message and DNA binding. Hypothalamic mRNA for AHR showed no change, while there was a 1.7-fold increase in DNA-binding activity. Our results show that in-utero and lactational exposure to TCDD alters the expression of AHR in a tissue-specific fashion, and that this altered regulation may either be a part or a cause of the general endocrine disruption observed in these animals.