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After birth, the development of secondary lymphoid tissues (SLTs) in the colon is dependent on the expression of the aryl hydrocarbon receptor (AhR) in immune cells as a response to the availability of AhR ligands. However, little is known about how AhR activity from intestinal epithelial cells (IECs) may influence the development of tertiary lymphoid tissues (TLTs). As organized structures that develop at sites of inflammation or infection during adulthood, TLTs serve as localized centers of adaptive immune responses, and their presence has been associated with the resolution of inflammation and tumorigenesis in the colon. Here, we investigated the effect of the conditional loss of AhR activity in IECs in the formation and immune cell composition of TLTs in a model of acute inflammation. In females, loss of AhR activity in IECs reduced the formation of TLTs without significantly changing disease outcomes or immune cell composition within TLTs. In males lacking AhR expression in IECs, increased disease activity index, lower expression of functional-IEC genes, increased number of TLTs, increased T-cell density, and lower B- to T-cell ratio were observed. These findings may represent an unfavorable prognosis when exposed to dextran sodium sulfate (DSS)-induced epithelial damage compared with females. Sex and loss of IEC AhR also resulted in changes in microbial populations in the gut. Collectively, these data suggest that the formation of TLTs in the colon is influenced by sex and AhR expression in IECs. NEW & NOTEWORTHY This is the first research of its kind to demonstrate a clear connection between biological sex and the development of tertiary lymphoid tissues (TLT) in the colon. In addition, the research finds that in a preclinical model of inflammatory bowel disease, the expression of the aryl hydrocarbon receptor (AhR) influences the development of these structures in a sex-specific manner.

Background and Objective: Despite that inherited (thalassemia and SCA) and infectious (hepatitis B, hepatitis C and AIDs) diseases and their risk factor consanguineous marriage are widespread among Yemen population, community-based awareness studies towards premarital screening (PMS) is extremely limited, so our study is designed to elicit knowledge and attitude of engaged and recently married couples in Taiz (Yemen) toward PMS. Methods: A cross-sectional study was conducted from April to September 2021 at Taiz. A well-structured questionnaire was designed and then delivered to participants to collect data. Results: The results revealed that majority of respondents (83.60%-94.18%) had knowledge about inherited and infectious diseases, respectively, and educational institution was the main knowledge source. Females had significantly higher knowledge than males ( P ≤ .01), except about AIDs since no difference ( P = .539). The results also revealed that majority of respondents had positive attitudes toward PMS, except toward AIDs testing since 70% of males and 68.91% of females had negative attitudes. The majority of respondents agreed that screening reduces the diseases incidence. Approximately half of respondents (50.79%-56.61%) tend to go ahead for marriage in case of positive results for inherited and infectious diseases respectively. Making PMS as an obligatory step prior engagement was agreed by 47.62% of respondents (45.71% of males, 48.74% of females). Conclusion: A high level of knowledge and positive attitudes toward inherited and infectious diseases and PMS were reported. This knowledge reflected on the agreement of majority of respondents regarding PMS importance in term of reduction of diseases incidence.

IL22 signaling plays an important role in maintaining gastrointestinal epithelial barrier function, cell proliferation, and protection of intestinal stem cells from genotoxicants. Emerging studies indicate that the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, promotes production of IL22 in gut immune cells. However, it remains to be determined if AhR signaling can also affect the responsiveness of colonic epithelial cells to IL22. Here, we show that IL22 treatment induces the phosphorylation of STAT3, inhibits colonic organoid growth, and promotes colonic cell proliferation in vivo. Notably, intestinal cell-specific AhR knockout (KO) reduces responsiveness to IL22 and compromises DNA damage response after exposure to carcinogen, in part due to the enhancement of suppressor of cytokine signaling 3 (SOCS3) expression. Deletion of SOCS3 increases levels of pSTAT3 in AhR KO organoids, and phenocopies the effects of IL22 treatment on wild-type (WT) organoid growth. In addition, pSTAT3 levels are inversely associated with increased azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon tumorigenesis in AhR KO mice. These findings indicate that AhR function is required for optimal IL22 signaling in colonic epithelial cells and provide rationale for targeting AhR as a means of reducing colon cancer risk. NEW & NOTEWORTHY AhR is a key transcription factor controlling expression of IL22 in gut immune cells. In this study, we show for the first time that AhR signaling also regulates IL22 response in colonic epithelial cells by modulating SOCS3 expression.

The mutational genetic landscape of colorectal cancer has been extensively characterized; however, the ability of "cooperation response genes" to modulate the function of cancer "driver" genes remains largely unknown. In this study, we investigate the role of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, in modulating oncogenic cues in the colon. We show that intestinal epithelial cell-targeted AhR knockout (KO) promotes the expansion and clonogenic capacity of colonic stem/progenitor cells harboring Apc S580/+ ; Kras G12D/+ mutations by upregulating Wnt signaling. The loss of AhR in the gut epithelium increased cell proliferation, reduced mouse survival rate, and promoted cecum and colon tumorigenesis in mice. Mechanistically, the antagonism of Wnt signaling induced by Lgr5 haploinsufficiency attenuated the effects of AhR KO on cecum and colon tumorigenesis. IMPLICATIONS: Our findings reveal that AhR signaling plays a protective role in genetically induced colon tumorigenesis at least by suppressing Wnt signaling and provides rationale for the AhR as a therapeutic target for cancer prevention and treatment., (©2021 American Association for Cancer Research.)

Background: Diet, loss of aryl hydrocarbon receptor (AhR) expression and their modification of the gut microbiota community composition and its metabolites affect the development of colorectal cancer (CRC). However, the concordance between fecal microbiota composition and the fecal metabolome is poorly understood. Mice with specific AhR deletion (AhRKO) in intestinal epithelial cell and their wild-type littermates were fed a low-fat diet or a high-fat diet. Shifts in the fecal microbiome and metabolome associated with diet and loss of AhR expression were assessed. Microbiome and metabolome data were integrated to identify specific microbial taxa that contributed to the observed metabolite shifts., Results: Our analysis shows that diet has a more pronounced effect on mouse fecal microbiota composition than the impact of the loss of AhR. In contrast, metabolomic analysis showed that the loss of AhR in intestinal epithelial cells had a more pronounced effect on metabolite profile compared to diet. Integration analysis of microbiome and metabolome identified unclassified Clostridiales, unclassified Desulfovibrionaceae, and Akkermansia as key contributors to the synthesis and/or utilization of tryptophan metabolites., Conclusions: Akkermansia are likely to contribute to the synthesis and/or degradation of tryptophan metabolites. Our study highlights the use of multi-omic analysis to investigate the relationship between the microbiome and metabolome and identifies possible taxa that can be targeted to manipulate the microbiome for CRC treatment.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that senses xenobiotics, diet, and gut microbial-derived metabolites, is increasingly recognized as a key regulator of intestinal biology. However, its effects on the function of colonic stem and progenitor cells remain largely unexplored. Here, we observed that inducible deletion of AhR in Lgr5 + stem cells increases the percentage of colonic stem cells and enhances organoid initiating capacity and growth of sorted stem and progenitor cells, while AhR activation has the opposite effect. Moreover, intestinal-specific AhR knockout increases basal stem cell and crypt injury-induced cell proliferation and promotes colon tumorigenesis in a preclinical colitis-associated tumor model by upregulating FoxM1 signaling. Mechanistically, AhR transcriptionally suppresses FoxM1 expression. Activation of AhR in human organoids recapitulates phenotypes observed in mice, such as reduction in the percentage of colonic stem cells, promotion of stem cell differentiation, and attenuation of FoxM1 signaling. These findings indicate that the AhR-FoxM1 axis, at least in part, mediates colonic stem/progenitor cell behavior., (© 2020 The Authors.)

Tumor metastasis to the draining lymph nodes is critical in patient prognosis and is tightly regulated by molecular interactions mediated by lymphatic endothelial cells (LECs). The underlying mechanisms remain undefined in the head and neck squamous cell carcinomas (HNSCCs). Using HNSCC cells and LECs we determined the mechanisms mediating tumor-lymphatic cross talk. The effects of a pentacyclic triterpenoid, methyl 2-trifluoromethyl-3,11-dioxoolean-1,12-dien-30-oate (CF 3 DODA-Me), a potent anticancer agent, were studied on cancer-lymphatic interactions. In response to inflammation, LECs induced the chemokine (C-X-C motif) ligand 9/10/11 chemokines with a concomitant increase in the chemokine (C-X-C motif) receptor 3 (CXCR3) in tumor cells. CF 3 DODA-Me showed antiproliferative effects on tumor cells, altered cellular bioenergetics, suppressed matrix metalloproteinases and chemokine receptors, and the induction of CXCL11-CXCR3 axis and phosphatidylinositol 3-kinase/AKT pathways. Tumor cell migration to LECs was inhibited by blocking CXCL11 whereas recombinant CXCL11 significantly induced tumor migration, epithelial-to-mesenchymal transition, and matrix remodeling. Immunohistochemical analysis of HNSCC tumor arrays showed enhanced expression of CXCR3 and increased lymphatic vessel infiltration. Furthermore, The Cancer Genome Atlas RNA-sequencing data from HNSCC patients also showed a positive correlation between CXCR3 expression and lymphovascular invasion. Collectively, our data suggest a novel mechanism for cross talk between the LECs and HNSCC tumors through the CXCR3-CXCL11 axis and elucidate the role of the triterpenoid CF 3 DODA-Me in abrogating several of these tumor-promoting pathways., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Consumption of a high-fat diet has been associated with an increased risk of developing colorectal cancer (CRC). However, the effects of the interaction between dietary fat content and the aryl hydrocarbon receptor (AhR) on colorectal carcinogenesis remain unclear. Mainly known for its role in xenobiotic metabolism, AhR has been identified as an important regulator for maintaining intestinal epithelial homeostasis. Although previous research using whole body AhR knockout mice has revealed an increased incidence of colon and cecal tumors, the unique role of AhR activity in intestinal epithelial cells (IECs) and modifying effects of fat content in the diet at different stages of sporadic CRC development are yet to be elucidated. In the present study, we have examined the effects of a high-fat diet on IEC-specific AhR knockout mice in a model of sporadic CRC. Although loss of AhR activity in IECs significantly induced the development of premalignant lesions, in a separate experiment, no significant changes in colon mass incidence were observed. Moreover, consumption of a high-fat diet promoted cell proliferation in crypts at the premalignant colon cancer lesion stage and colon mass multiplicity as well as β-catenin expression and nuclear localization in actively proliferating cells in colon masses. Our data demonstrate the modifying effects of high-fat diet and AhR deletion in IECs on tumor initiation and progression. NEW & NOTEWORTHY Through the use of an intestinal-specific aryl hydrocarbon receptor (AhR) knockout mouse model, this study demonstrates that the expression of AhR in intestinal epithelial cells is required to reduce the formation of premalignant colon cancer lesions. Furthermore, consumption of a high-fat diet and the loss of AhR in intestinal epithelial cells influences the development of colorectal cancer at various stages.

3,3'-Diindolylmethane (DIM) and its synthetic halogenated derivatives 4,4'-Br 2 - and 7,7'-Cl 2 DIM (ring-DIMs) have recently been shown to induce protective autophagy in human prostate cancer cells. The mechanisms by which DIM and ring-DIMs induce autophagy have not been elucidated. As DIM is a mitochondrial ATP-synthase inhibitor, we hypothesized that DIM and ring-DIMs induce autophagy via alteration of intracellular AMP/ATP ratios and activation of AMP-activated protein kinase (AMPK) signaling in prostate cancer cells. We found that DIM and ring-DIMs induced autophagy was accompanied by increased autophagic vacuole formation and conversion of LC3BI to LC3BII in LNCaP and C42B human prostate cancer cells. DIM and ring-DIMs also induced AMPK, ULK-1 (unc-51-like autophagy activating kinase 1; Atg1) and acetyl-CoA carboxylase (ACC) phosphorylation in a time-dependent manner. DIM and the ring-DIMs time-dependently induced the oncogenic protein astrocyte-elevated gene 1 (AEG-1) in LNCaP and C42B cells. Downregulation of AEG-1 or AMPK inhibited DIM- and ring-DIM-induced autophagy. Pretreatment with ULK1 inhibitor MRT 67307 or siRNAs targeting either AEG-1 or AMPK potentiated the cytotoxicity of DIM and ring-DIMs. Interestingly, downregulation of AEG-1 induced senescence in cells treated with overtly cytotoxic concentrations of DIM or ring-DIMs and inhibited the onset of apoptosis in response to these compounds. In summary, we have identified a novel mechanism for DIM- and ring-DIM-induced protective autophagy, via induction of AEG-1 and subsequent activation of AMPK. Our findings could facilitate the development of novel drug therapies for prostate cancer that include selective autophagy inhibitors as adjuvants., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The present study demonstrates the promising anticancer effects of novel C-substituted diindolylmethane (DIM) derivatives DIM-10 and DIM-14 in aggressive TNBC models. In vitro studies demonstrated that these compounds possess strong anticancer effects. Caco-2 permeability studies resulted in poor permeability and poor oral bioavailability was demonstrated by pharmacokinetic studies. Nano structured lipid carrier (NLC) formulations were prepared to increase the clinical acceptance of these compounds. Significant increase in oral bioavailability was observed with NLC formulations. Compared to DIM-10, DIM-10 NLC formulation showed increase in C max and AUC values by 4.73 and 11.19-folds, respectively. Similar pattern of increase was observed with DIM-14 NLC formulations. In dogs DIM-10 NLC formulations showed an increase of 2.65 and 2.94-fold in C max and AUC, respectively. The anticancer studies in MDA-MB-231 orthotopic TNBC models demonstrated significant reduction in tumor volumes in DIM-10 and DIM-14 NLC treated animals. Our studies suggest that NLC formulation of both DIM-10 and 14 is effective in TNBC models., (Copyright © 2016. Published by Elsevier B.V.)

The genotoxicity of a complex mixture [neutral fraction (NF)] from a wood preserving waste and reconstituted mixture (RM) mimicking the NF with seven major polycyclic aromatic hydrocarbons (PAHs) and benzo(a)pyrene (BaP) was investigated by determining DNA adducts and tumor incidence in male B6C3F1 mice exposed to three different doses of the chemical mixtures. The peak values of DNA adducts were observed after 24 h, and the highest levels of PAH-DNA adducts were exhibited in mice administered NF + BaP, and the highest tumor incidence and mortality were also observed in this group. DNA adduct levels after 1, 7, or 21 days were significantly correlated with animal mortality and incidence of total tumors including liver, lung, and forestomach. However, only hepatic DNA adducts after 7 days significantly correlated with liver tumor incidence. Most proteins involved in DNA repair including ATM, pATR, Chk1, pChk1, DNA PKcs, XRCC1, FANCD2, Ku80, Mre11, and Brca2 were significantly lower in liver tumor tissue compared to non-tumor tissue. Expressions of proteins involved in apoptosis and cell cycle regulation were also significantly different in tumor versus non-tumor tissues, and it is possible that PAH-induced changes in these gene products are important for tumor development and growth.

We recently reported that novel ring-substituted analogs of 3,3'-diindolylmethane (ring-DIMs) induce apoptosis and necrosis in androgen-dependent and -independent prostate cancer cells. In this paper, we have focused on the mechanism(s) associated with ring-DIM-mediated cell death, and on identifying the specific intracellular target(s) of these compounds. The 4,4'- and 7,7'-dichloroDIMs and 4,4'- and 7,7'-dibromoDIMs induced the death of LNCaP, C42B and DU145 prostate cancer cells, but not that of immortalized normal human prostate epithelial (RWPE-1) cells. Ring-DIMs caused the early loss of mitochondrial membrane potential (MMP) and decreased mitochondrial ATP generation in prostate cancer cells. Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore, inhibited ring-DIM-mediated cell death, and salubrinal, an inhibitor of ER stress, inhibited cell death mediated only by 4,4'-dihaloDIMs. We found that although salubrinal did not inhibit the onset of ER stress, it prevented 4,4'-dibromoDIM mediated loss of MMP. Salubrinal potentiated cell death in response to 7,7'-dihaloDIMs and DIM, and this effect concurred with increased loss of MMP. Using in silico 3-D docking affinity analysis, we identified Ca2+/calmodulin-dependent kinase II (CaMKII) as a potential direct target for the most toxic ring-DIM, 4,4'-dibromoDIM. An inhibitor of CaMKII, KN93, but not its inactive analog KN92, abrogated cell death mediated by 4,4'-dibromoDIM. The ring-DIMs induced ER stress and autophagy, but these processes were not necessary for ring-DIM-mediated cell death. Inhibition of autophagy with bafilomycin A1, 3-methyladenine or by LC3B gene silencing sensitized LNCaP and C42B, but not ATG5-deficient DU145 cells to ring-DIM- and DIM-mediated cell death. We propose that autophagy induced by the ring-DIMs and DIM has a cytoprotective function in prostate cancer cells.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Triterpenoids such as betulinic acid (BA) and synthetic analogs of oleanolic acid [2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)] and glycyrrhetinic acid [2-cyano-3,11-dioxo-18β-oleana-1,12-dien-30-oc acid (CDODA)] are potent anticancer agents that exhibit antiproliferative, antiangiogenic, anti-inflammatory and pro-apoptotic activities. Although their effects on multiple pathways have been reported, unifying mechanisms of action have not been reported. Studies in this laboratory have now demonstrated that several triterpenoids including BA and some derivatives, celastrol, methyl ursolate, β-boswellic acid derivatives, and the synthetic analogs CDDO, CDODA and their esters decreased expression of specificity protein (Sp) transcription factors and several pro-oncogenic Sp-regulated genes in multiple cancer cell lines. The mechanisms of this response are both compound- and cell context-dependent and include activation of both proteasome-dependent and -independent pathways. Triterpenoid-mediated induction of reactive oxygen species (ROS) has now been characterized as an important proteasome-independent pathway for downregulation of Sp transcription factors. ROS decreases expression of microRNA-27a (miR-27a) and miR-20a/miR-17-5p and this results in the induction of the transcriptional "Sp-repressors" ZBTB10 and ZBTB4, respectively, which in turn downregulate Sp and Sp-regulated genes. Triterpenoids also activate or deactive nuclear receptors and G-protein coupled receptors, and these pathways contribute to their antitumorigenic activity and may also play a role in targeting Sp1, Sp3 and Sp4 which are highly overexpressed in multiple cancers and appear to be important for maintaining the cancer phenotype.

Aim: We hypothesized that the anticancer activity of cannabinoids was linked to induction of phosphatases., Materials and Methods: The effects of cannabidiol (CBD) and the synthetic cannabinoid WIN-55,212 (WIN) on LNCaP (prostate) and SW480 (colon) cancer cell proliferation were determined by cell counting; apoptosis was determined by cleavage of poly(ADP)ribose polymerase (PARP) and caspase-3 (Western blots); and phosphatase mRNAs were determined by real-time PCR. The role of phosphatases and cannabinoid receptors in mediating CBD- and WIN-induced apoptosis was determined by inhibition and receptor knockdown., Results: CBD and WIN inhibited LNCaP and SW480 cell growth and induced mRNA expression of several phosphatases, and the phosphatase inhibitor sodium orthovanadate significantly inhibited cannabinoid-induced PARP cleavage in both cell lines, whereas only CBD-induced apoptosis was CB1 and CB2 receptor-dependent., Conclusion: Cannabinoid receptor agonists induce phosphatases and phosphatase-dependent apoptosis in cancer cell lines; however, the role of the CB receptor in mediating this response is ligand-dependent.

Although rapid, membrane-activated estrogen receptor (ER) signaling is no longer controversial, the biological function of this nongenomic signaling is not fully characterized. We found that rapid signaling from membrane-associated ER regulates the histone methyltransferase enhancer of Zeste homolog 2 (EZH2). In response to both 17beta-estradiol (E2) and the xenoestrogen diethylstilbestrol, ER signaling via phosphatidylinositol 3-kinase/protein kinase B phosphorylates EZH2 at S21, reducing levels of trimethylation of lysine 27 on histone H3 in hormone-responsive cells. During windows of uterine development that are susceptible to developmental reprogramming, activation of this ER signaling pathway by diethylstilbestrol resulted in phosphorylation of EZH2 and reduced levels of trimethylation of lysine 27 on histone H3 in chromatin of the developing uterus. Furthermore, activation of nongenomic signaling reprogrammed the expression profile of estrogen-responsive genes in uterine myometrial cells, suggesting this as a potential mechanism for developmental reprogramming caused by early-life exposure to xenoestrogens. These data demonstrate that rapid ER signaling provides a direct linkage between xenoestrogen-induced nuclear hormone receptor signaling and modulation of the epigenetic machinery during tissue development.

We recently reported that colored potato extracts and an anthocyanin rich fraction suppressed lymph-node carcinoma of the prostate (LNCaP) and prostate cancer-3 (PC-3) prostate cancer cell proliferation and induced apoptosis via caspase-dependent and caspase-independent pathways. Chlorogenic acid, caffeic acid, gallic acid, catechin, malvidin, and glycoalkaloids (alpha-chaconine and solanine) have now been identified as the major bioactive components of potato, and their effects on LNCaP and PC-3 cell proliferation and apoptosis have been investigated. alpha-chaconine (5 microg/ml) and gallic acid (15 microg/ml) exhibited potent antiproliferative properties and increased cyclin-dependent kinase inhibitor p27 levels in both cell lines. Both alpha-chaconine and gallic acid induced poly [adenosine diphosphate (ADP)] ribose polymerase cleavage and caspase-dependent apoptosis in LNCaP cells; however, caspase-independent apoptosis through nuclear translocation of endonuclease G was observed in both LNCaP and PC-3 cells. alpha-chaconine and gallic acid activated c-Jun N-terminal protein kinase (JNK), and this response played a major role in induction of caspase-dependent apoptosis in LNCaP cells; whereas modulation of JNK and mitogen-activated protein kinase did not affect alpha-chaconine- and gallic acid-induced caspase-independent apoptosis. These results suggest that apoptosis induced by whole potato extracts in prostate cancer cell lines may be in part due to alpha-chaconine and gallic acid.

Polyphenols from fruits and vegetables exhibit anticancer properties both in vitro and in vivo and specialty potatoes are an excellent source of dietary polyphenols, including phenolic acids and anthocyanins. This study investigated the effects of specialty potato phenolics and their fractions on LNCaP (androgen dependent) and PC-3 (androgen independent) prostate cancer cells. Phenolic extracts from four specialty potato cultivars CO112F2-2, PATX99P32-2, ATTX98462-3 and ATTX98491-3 and organic acid, phenolic acid and anthocyanin fractions (AF) were used in this study. CO112F2-2 cultivar extracts and their AF at 5 mug chlorogenic acid eq/ml were more active and inhibited cell proliferation and increased the cyclin-dependent kinase inhibitor p27 levels in both LNCaP and PC-3 cells. Potato extract and AF induced apoptosis in both the cells and, however, the effects were cell context dependent. Cell death pathways induced by potato extract and AF were associated with mitogen-activated protein kinase and c-jun N-terminal kinase activation and these kinases activated caspase-independent apoptosis through nuclear translocation of endonuclease G (Endo G) and apoptosis-inducing factor in both cell lines. Induction of caspase-dependent apoptosis was also kinase dependent but was observed only in LNCaP cells. Kinase inhibitors reversed this nuclear translocation of endonuclease G and apoptosis-inducing factor. This is the first report showing that the cytotoxic activities of potato extract/AF in cancer cells were due to activation of caspase-independent apoptosis. Current studies are focused on identifying individual components of the AF responsible for the induction of cell death pathways in prostate and other cancer cell lines and developing potato cultivars that overexpress these active compounds.

At the request of the U.S. Environmental Protection Agency (EPA) Office of Research and Development, a subcommittee of the Board of Scientific Counselors Executive Committee conducted an independent and open peer review of the Endocrine Disrupting Chemicals Research Program (EDC Research Program) of the U.S. EPA. The subcommittee was charged with reviewing the design, relevance, progress, scientific leadership, and resources of the program. The subcommittee found that the long-term goals and science questions in the EDC Program are appropriate and represent an understandable and solid framework for setting research priorities, representing a combination of problem-driven and core research. Long-term goal (LTG) 1, dealing with the underlying science surrounding endocrine disruptors, provides a solid scientific foundation for conducting risk assessments and making risk management decisions. LTG 2, dealing with defining the extent of the impact of endocrine-disrupting chemicals (EDCs), has shown greater progress on ecologic effects of EDCs compared with that on human health effects. LTG 3, which involves support of the Endocrine Disruptor Screening and Testing Program of the U.S. EPA, has two mammalian tests already through a validation program and soon available for use. Despite good progress, we recommend that the U.S. EPA a) strengthen their expertise in wildlife toxicology, b) expedite validation of the Endocrine Disruptors Screening and Testing Advisory Committee tests, c) continue dependable funding for the EDC Research Program, d) take a leadership role in the application of "omics" technologies to address many of the science questions critical for evaluating environmental and human health effects of EDCs, and e) continue to sponsor multidisciplinary intramural research and interagency collaborations.

HER2 overexpression is one of the most recognizable molecular alterations in breast tumors known to be associated with a poor prognosis. In the study described here, we explored the effect of HER2 overexpression on the sensitivity of breast cancer cells to the growth-inhibitory effects of 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), a synthetic triterpenoid, both in vitro and in vivo in a xenograft model of breast cancer. Both cell growth and colony formation in the soft agar assay, a hallmark of the transformation phenotype, were preferentially suppressed in HER2-overexpressing cell lines at low concentrations of CDDO, whereas growth-inhibitory effects at high concentrations did not correlate with the expression level of HER2. CDDO dose-dependently inhibited phosphorylation of HER2 in HER2-overexpressing cells and diminished HER2 kinase activity in vitro. CDDO induced the transactivation of the nuclear receptor peroxisome proliferator-activated receptor-gamma in both vector control and HER2-transfected MCF7 cells. Dose-response studies showed that the growth inhibition seen at lower concentrations of CDDO correlated with induction of the tumor suppressor gene caveolin-1, which is known to inhibit breast cancer cell growth. CDDO also reduced cyclin D1 mRNA and protein expression. In vivo studies with liposomally encapsulated CDDO showed complete abrogation of the growth of the highly tumorigenic MCF7/HER2 cells in a xenograft model of breast cancer. These findings provide the first in vitro and in vivo evidence that CDDO effectively inhibits HER2 tyrosine kinase activity and potently suppresses the growth of HER2-overexpressing breast cancer cells and suggest that CDDO has a therapeutic potential in advanced breast cancer.

Establishment of pregnancy in ruminants results from paracrine signaling by interferon tau (IFNT) from the conceptus to uterine endometrial luminal epithelia (LE) that prevents release of luteolytic prostaglandin F(2alpha) pulses. In cyclic and pregnant ewes, progesterone down-regulates progesterone receptor (PGR) gene expression in LE. In cyclic ewes, loss of PGR allows for increases in estrogen receptor alpha (ESR1) and then oxytocin receptor (OXTR) gene expression followed by oxytocin-induced prostaglandin F(2alpha) pulses. In pregnant ewes, IFNT inhibits transcription of the ESR1 gene, which presumably inhibits OXTR gene transcription. Alternatively, IFNT may directly inhibit OXTR gene transcription. The 5' promoter/enhancer region of the ovine OXTR gene was cloned and found to contain predicted binding sites for activator protein 1, SP1, and PGR, but not for ESR1. Deletion analysis showed that the basal promoter activity was dependent on the region from -144 to -4 bp that contained only SP1 sites. IFNT did not affect activity of the OXTR promoter. In cells transfected with ESR1, E2, and ICI 182,780 increased promoter activity due to GC-rich SP1 binding sites at positions -104 and -64. Mutation analyses showed that the proximal SP1 sites mediated ESR1 action as well as basal activity of the promoter. In response to progesterone, progesterone receptor B also increased OXTR promoter activity. SP1 protein was constitutively expressed and abundant in the LE of the ovine uterus. These results support the hypothesis that the antiluteolytic effects of IFNT are mediated by direct inhibition or silencing of ESR1 gene transcription, thereby precluding ESR1/SP1 from stimulating OXTR gene transcription.

Insulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that induces proliferation of MCF-7 breast cancer cells, and cotreatment with the phosphoinositide 3-kinase (PI3-K) inhibitor LY294002 and the antiestrogen ICI 182780 inhibits IGF-I-induced growth. The role of estrogen receptor alpha (ERalpha) in mediating responses induced by IGF-I was investigated in cells transfected with small inhibitory RNA for ERalpha (iERalpha). The results showed that IGF-I-dependent phosphorylation of Akt and mitogen-activated protein kinase, induction of G(1)-S-phase progression and enhanced expression of cyclin D1 and cyclin E were dependent on ERalpha. Moreover, these same IGF-I-induced responses were also inhibited by the antiestrogen ICI 182780 and this was in contrast to a previous report suggesting that ICI 182780 did not affect IGF-I-dependent activation of PI3-K or induction of cyclin D1 expression. ICI 182780 exhibits antimitogenic activity and iERalpha inhibits G(1)-S-phase progression and proliferation of MCF-7 cells treated with IGF-I, suggesting that the effects of the antiestrogen are primarily related to downregulation of ERalpha.

Proinflammatory cytokines and adhesion molecules expressed by endothelial cells (ECs) play a critical role in initiating and promoting atherosclerosis. Agents that oppose these inflammatory effects in vascular cells include peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands, including 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and synthetic thiazolidinediones. Recently, a new structural class of potent PPAR-gamma agonists, 1,1-bis(3'-indolyl)-1-(p-substituted phenyl) methanes, has been characterized. The purpose of this study was to evaluate the anti-inflammatory effects of two PPAR-gamma-active members of this class, 1,1-bis(3'-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu) and 1,1-bis(3'-indolyl)-1-(p-biphenyl)methane (DIM-C-pPhC(6)H(5)), in ECs in vitro. Pretreatment of ECs with DIM-C-pPhC(6)H(5), DIM-C- pPhtBu, or 15d-PGJ2 decreased tumor necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule (ICAM)-1 expression in a concentration-dependent manner. At a concentration of 10 microM, DIM-C-pPhtBu and DIM-C-pPhC(6)H(5) decreased ICAM-1 expression by 77.5 and 71.3%, respectively, and comparable inhibition (84.4%) was observed for 10 microM 15d-PGJ2 (p < 0.05). In contrast, 10 microM ciglitazone and DIM-C-pPhCH(3), which exhibits low PPAR-gamma agonist activity, were inactive. The two new PPAR-gamma agonists and 15d-PGJ2 also inhibited TNF-alpha-induced interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production in supernatants of TNF-alpha-stimulated ECs, whereas ciglitazone and DIM-C-pPhCH(3) did not decrease TNF-alpha-induced expression of these two proteins. This new structural class of PPAR-gamma agonists inhibited the expression of ICAM-1 and the production of IL-6 and MCP-1 in TNF-alpha-activated ECs at lower concentrations than other synthetic PPAR-gamma agonists, suggesting the potential clinical utility of 1,1-bis(3'-indolyl)-1-(p-substituted phenyl) methanes for decreasing endothelial inflammation.

We investigated the antileukemic activity and molecular mechanisms of action of a newly synthesized ring-substituted diindolylmethane derivative, 1,1-bis[3'-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), in acute myelogenous leukemia (AML) cells. DIM #34 inhibited AML cell growth via the induction of apoptosis and abrogated clonogenic growth of primary AML samples. Exposure to DIM #34 induced loss of mitochondrial inner transmembrane potential, release of cytochrome c into the cytosol, and caspase activation. Bcl-2-overexpressing, Bax knockout, and caspase-9-deficient cells were partially resistant to cell death, suggesting the involvement of the intrinsic apoptotic pathway. Furthermore, DIM #34 transiently inhibited the phosphorylation and activity of the extracellular signal-regulated kinase and abrogated Bcl-2 phosphorylation. Because other methylene-substituted diindolylmethane analogues have been shown to transactivate the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma), we studied the role of PPARgamma in apoptosis induction. Cotreatment of cells with a selective PPARgamma antagonist or with retinoid X receptor and retinoic acid receptor ligands partially modulated apoptosis when combined with DIM #34, suggesting PPARgamma receptor-dependent and receptor-independent cell death. Together, these findings suggest that diindolylmethanes are a new class of compounds that selectively induce apoptosis in AML cells through the modulation of the extracellular signal-regulated kinase and PPARgamma signaling pathways.

Polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) are known to bind to transthyretin (TTR) in vitro, possibly explaining their bioaccumulation, rodent toxicity, and presumed human toxicity. Herein, we show that several OH-PCBs bind selectively to TTR in blood plasma; however, only one of the PCBs tested binds TTR in plasma. Some of the OH-PCBs displace thyroid hormone (T4) from TTR, rationalizing the toxicity observed in rodents, where TTR is the major T4 transporter. Thyroid binding globulin and albumin are the major T4 carriers in humans, making it unlikely that enough T4 could be displaced from TTR to be toxic. OH-PCBs are excellent TTR amyloidogenesis inhibitors in vitro because they bind to the TTR tetramer, imparting kinetic stability under amyloidogenic denaturing conditions. Four OH-PCB/TTR cocrystal structures provide further insight into inhibitor binding interactions.

1. The naturally occurring compounds curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), 8-prenylnaringenin (8PN), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) protect animals against chemically induced tumours. Putative chemoprotective mechanisms include modulated expression of hepatic biotransformation enzymes. However, few, if any, studies have used human primary cells as test models. 2. The present study investigated the effects of these phytochemicals on the expression of four carcinogenesis-relevant enzymes--cytochrome P450 (CYP)1A1 and 1A2, NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferase A1 (GSTA1)--in primary cultures of freshly isolated human hepatocytes. 3. Quantitative RT-PCR analyses demonstrated that CYP1A1 was up-regulated by PEITC and DIM in a dose-dependent manner. CYP1A2 transcription was significantly activated following DIM, IXN, 8PN and PEITC treatments. DIM exhibited a remarkably effective induction response of CYP1A1 (474-, 239- and 87-fold at 50, 25 and 10 microM, respectively) and CYP1A2 (113-, 70- and 31-fold at 50, 25 and 10 microM, respectively), that was semiquantitatively reflected in protein levels. NQO1 expression responded to PEITC (11 x at 25 microM), DIM (4.5 x at 50 microM) and SFN (5 x at 10 microM) treatments. No significant effects on GSTA1 transcription were seen. 4. The findings show novel and unexpected effects of these phytochemicals on the expression of human hepatic biotransformation enzymes that play key roles in chemical-induced carcinogenesis.

Manufactured gas plant residue (MGP) is a complex mixture of polycyclic aromatic hydrocarbons (PAHs) that is tumorigenic in the lungs of mice. This study compared the relative genotoxicity of 7H-benzo[c]fluorene (BC), a PAH component of MGP, with MGP and MGP fractions in order to assess the contribution of BC to the genotoxicity of MGP. An MGP sample was separated into seven fractions (F1-F7) using silica gel column chromatography with petroleum ether (PE) followed by PE:acetone (99:1 v/v, then 98:2). PAHs were quantified using gas chromatography/mass spectrometry. An aliquot of F2, the fraction with the highest BC concentration and highest weighted mutagenic activity in Salmonella typhimurium strain TA98, was further separated using silica gel thin-layer chromatography with hexane. The first F2 subfraction, sF2-a, was enriched in BC and coeluting compounds and contained 35,000 ppm BC and 216,109 ppm carcinogenic PAHs (cPAHs, the sum of seven PAHs categorized by the U.S. EPA as class B2 carcinogens). The second F2 subfraction, sF2-b, contained a ninefold lower concentration of BC, with 3,900 ppm BC and 45,216 ppm cPAHs. Female ICR mice received topical application of crude MGP, crude MGP spiked with analytical-grade BC, F2, sF2-a, sF2-b, or analytical-grade BC. DNA adduct levels were analyzed by nuclease P1-enhanced (32)P-postlabeling. In lung DNA of mice receiving 0.48 or 3.0 mg/mouse, net total RAL x 10(9) values were F2, 30.8 and 87.2; sF2-a, 24.8 and 106.7; and sF2-b, 19.6 and 151.0, respectively. Mice dosed with 0.10 mg analytical-grade BC (the mass of BC in 3.0 mg sF2-a) exhibited a net total RAL x 10(9) value of 7.03 in lung DNA. This was equal to approximately 7% of the total RAL x 10(9) value produced by 3.0 mg sF2-a. Thus, although BC appears to make an appreciable contribution to pulmonary adduct formation, the results suggest that MGP components other than BC play an important role in lung DNA adduct formation following topical MGP administration., (Copyright 2004 Wiley-Liss, Inc.)

Chemoprotective phytochemicals exhibit multiple activities and interact with several cellular receptors, including the aryl hydrocarbon (Ah) receptor (AhR). In this study we investigated the AhR agonist/antagonist activities of the following flavonoids: chrysin, phloretin, kaempferol, galangin, naringenin, genistein, quercetin, myricetin, luteolin, baicalein, daidzein, apigenin, and diosmin. We also investigated the AhR-dependent activities of cantharidin and emodin (in herbal extracts) in Ah-responsive MCF-7 human breast cells, HepG2 human liver cancer cells, and mouse Hepa-1 cells transiently or stably transfected with plasmids expressing a luciferase reporter gene linked to multiple copies of a consensus dioxin-responsive element. The AhR agonist activities of the compounds (1 and 10 micro M) were as high as 25% of the maximal response induced by 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and their potencies were dependent on cell context. Galangin, genistein, daidzein, and diosmin were active only in Hepa-1 cells, and cantharidin induced activity only in human HepG2 and MCF-7 cells. Western blot analysis confirmed that baicalein and emodin also induced CYP1A1 protein in the human cancer cell lines. The AhR antagonist activities of four compounds inactive as agonists in MCF-7 and HepG2 cells (kaempferol, quercetin, myricetin, and luteolin) were also investigated. Luteolin was an AhR antagonist in both cell lines, and the inhibitory effects of the other compound were dependent on cell context. These data suggest that dietary phytochemicals exhibit substantial cell context-dependent AhR agonist as well as antagonist activities. Moreover, because phytochemicals and other AhR-active compounds in food are present in the diet at relatively high concentrations, risk assessment of dietary toxic equivalents of TCDD and related compounds should also take into account AhR agonist/antagonist activities of phytochemicals.

Estrogenic industrial compounds such as bisphenol A (BPA) and nonylphenol typically bind estrogen receptor (ER) alpha and ERBeta and induce transactivation of estrogen-responsive genes/reporter genes, but their potencies are usually greater than or equal to 1,000-fold lower than observed for 17Beta-estradiol. Risk assessment of estrogenic compounds on the basis of their potencies in simple reporter gene or binding assays may be inappropriate. For example, selective ER modulators (SERMs) represent another class of synthetic estrogens being developed for treatment of hormone-dependent problems. SERMs differentially activate wild-type ERalpha and variant forms expressing activation function 1 (ER-AF1) and AF2 (ER-AF2) in human HepG2 hepatoma cells transfected with an estrogen-responsive complement C3 promoter-luciferase construct, and these in vitro differences reflect their unique in vivo biologies. The HepG2 cell assay has also been used in our laboratories to investigate the estrogenic activities of the following structurally diverse synthetic and phytoestrogens: 4 -hydroxytamoxifen; BPA; 2 ,4 ,6 -trichloro-4-biphenylol; 2 ,3 ,4 ,5 -tetrachloro-4-biphenylol; p-t-octylphenol; p-nonylphenol; naringenin; kepone; resveratrol; and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane. The results show that synthetic and phytoestrogens are weakly estrogenic but induce distinct patterns of ER agonist/antagonist activities that are cell context- and promoter-dependent, suggesting that these compounds will induce tissue-specific (in vivo(ER agonist or antagonist activities. These results suggest that other receptors, such as the aryl hydrocarbon receptor, that also bind structurally diverse ligands may exhibit unique responses in vivo that are not predicted by standard in vitro bioassays.

Intracellular Ca2+ oscillations induced by oxytocin and vasopressin were analyzed in a rat liver cell line (Clone 9) in order to identify mechanisms by which benzo[a]pyrene (BaP) alters Ca2+ signaling patterns in these cells. Clone 9 cells exhibit an initial Ca2+ spike, followed by Ca2+ oscillations upon oxytocin or vasopressin treatment. The range of frequencies (maximum 110 mHz) was dependent on agonist concentration with a constant amplitude less than or equal to the amount of Ca2+ generated from the inositol trisphosphate (InsP(3))-sensitive pool. This study examined contributions of extracellular and intracellular pools to the frequency of Ca2+ oscillations and the role of membrane channels, second messengers, and different pharmacological reagents on the regulation of oscillation frequency in both control and BaP-treated cells. Results indicated that the Ca2+ oscillations are mainly due to inositol 1,4,5-triphosphate (InsP(3))-sensitive stores and that extracellular Ca2+ contributes to refilling of this intracellular Ca2+ pool. The frequency of Ca2+ oscillations is also sharply affected by protein kinase C activated by phospholipase C. In BaP-treated Clone 9 cells, basal Ca2+ levels were elevated and the frequency of Ca2+ oscillations was suppressed in a dose-dependent fashion. Suppression of Ca2+ oscillations is due, at least in part, to an effect of BaP on enhanced opening of K+ channels. This was confirmed by showing that inhibition of the K+ channel opening by tetraethylammonium chloride can reverse the effect of BaP on oxytocin-induced Ca2+ oscillations, and potentially decrease the toxicity of BaP.

Diindolylmethane (DIM) is formed by acid catalyzed dimerization of the phytochemical indole-3-carbinol, and both compounds inhibit formation and/or growth of mammary tumors in rodents. In this study, we have investigated the aryl hydrocarbon receptor (AhR) agonist activity and inhibitory AhR-estrogen receptor crosstalk induced by the following methyl-substituted DIMs: 1,1'-dimethyl-, 2,2'-dimethyl-, 5,5'-dimethyl-, 6,6'-dimethyl-, and 7,7'-dimethylDIM and 1,1',2,2'-tetramethylDIM. The six compounds bound to the rat cytosolic AhR in a transformation assay but, at concentrations < or = 10 microM, exhibited minimal to non-detectable AhR agonist or antagonist activities associated with CYP1A1 induction. In contrast, the methyl-substituted DIMs inhibited estrogen-induced T47D human breast cancer cell growth and the four most active compounds (1,1'-, 2,2'-, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM) inhibited one or more estrogen-induced responses in the 21-day-old female B6C3F1 mice at a dose of 100 mg/kg/day (X3). Induction of hepatic CYP1A1-dependent activity was not observed at this high dose. The antitumorigenic activity of these compounds was examined in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor model in which the DIM analogs were orally administered (by gavage in corn oil) at a dose of 1 mg/kg/day (X10). 1,1'-DimethylDIM, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM significantly inhibited mammary tumor growth, and this was not accompanied by changes in organ/body weights or histopathology. These studies demonstrate that methyl-substituted DIMs are selective AhR modulators (SAhRMs) with potential for clinical treatment of breast cancer.

It has been hypothesized that environmental contaminants that modulate endocrine signaling pathways may be causally linked to adverse health effects in humans. There has been particular concern regarding synthetic estrogens and their role in disrupting normal development of the male reproductive tract. Most estrogenic industrial compounds, such as bisphenol A (BPA) and nonylphenol, typically bind estrogen receptors alpha (ERalpha) and beta (ERbeta) and induce transactivation of estrogen-responsive genes/reporter genes, but their potencies are usually > or = 1,000-fold lower than observed for 17beta-estradiol (E2). Selective estrogen receptor modulators (SERMs) represent another class of synthetic estrogens that are being developed for treatment of hormone-dependent problems. The SERMs differentially activate wild-type ERalpha and variant forms expressing activation function 1 (ER-AF1) and AF2 (ER-AF2) in human HepG2 hepatoma cells transfected with a pC3-luciferase construct, and these in vitro differences reflect their unique in vivo biologies. The HepG2 cell assay has also been used in our laboratories to investigate the estrogenic activities of the following structurally diverse synthetic and phytoestrogens: 4'-hydroxytamoxifen; BPA; 2',4',6'-trichloro-4-biphenylol; 2',3',4',5'-tetrachloro-4-biphenylol; p-t-octylphenol; p-nonylphenol; naringenin; kepone; resveratrol; and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). The results show that synthetic and phytoestrogens induce distinct patterns of gene activation in HepG2 and U2 osteogenic sarcoma cells, suggesting that these compounds will induce tissue-specific in vivo ER agonist or antagonist activities. The predicted differences between these compounds, based on results of the in vitro bioassay, have been confirmed. For example, BPA inhibits E2-induced responses in the rodent uterus, and HPTE and structurally related compounds are ERalpha agonists and ERbeta antagonists in assays carried out in HepG2 and other cancer cell lines.

Interferon-tau (IFNtau), a type I IFN produced by sheep conceptus trophectoderm, is the signal for maternal recognition of pregnancy. Although it is clear that IFNtau suppresses transcription of the estrogen receptor alpha and oxytocin receptor genes and induces expression of various IFN-stimulated genes within the endometrial epithelium, little is known of the signal transduction pathway activated by the hormone. This study determined the effects of IFNtau on signal transducer and activator of transcription (STAT) activation, expression, DNA binding, and transcriptional activation using an ovine endometrial epithelial cell line. IFNtau induced persistent tyrosine phosphorylation and nuclear translocation of STAT1 and -2 (10 min to 48 h), but transient phosphorylation and nuclear translocation of STAT3, -5a/b, and -6 (10 to <60 min). IFNtau increased expression of STAT1 and -2, but not STAT3, -5a/b, and -6. IFN-stimulated gene factor-3 and STAT1 homodimers formed and bound an IFN-stimulated response element (ISRE) and gamma-activated sequence (GAS) element, respectively. IFNtau increased transcription of GAS-driven promoters at 3 h, but suppressed their activity at 24 h. In contrast, the activity of an ISRE-driven promoter was increased at 3 and 24 h. These results indicate that IFNtau activates multiple STATs and has differential effects on ISRE- and GAS-driven gene transcription.

It has been hypothesized that environmental exposure to synthetic estrogenic chemicals and related endocrine-active compounds may be responsible for a global decrease in sperm counts, decreased male reproductive capacity, and breast cancer in women. Results of recent studies show that there are large demographic variations in sperm counts within countries or regions, and analyses of North American data show that sperm counts have not decreased over the last 60 years. Analyses of records for hypospadias and cryptorchidism also show demographic differences in these disorders before 1985; however, since 1985 rates of hypospadias have not changed and cryptorchidism has actually declined. Temporal changes in sex ratios and fertility are minimal, whereas testicular cancer is increasing in most countries; however, in Scandinavia, the difference between high (Denmark) and low (Finland) incidence areas are not well understood and are unlikely to be correlated with differences in exposure to synthetic industrial chemicals. Results from studies on organochlorine contaminants (DDE/PCB) show that levels were not significantly different in breast cancer patients versus controls. Thus, many of the male and female reproductive tract problems linked to the endocrine-disruptor hypothesis have not increased and are not correlated with synthetic industrial contaminants. This does not exclude an endocrine-etiology for some adverse environmental effects or human problems associated with high exposures to some chemicals.

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants which exert a variety of toxic effects in animals, including disturbances of sexual development and reproductive function. The estrogenic effects of PCBs may be mediated in part by hydroxylated PCB metabolites (PCB-OHs), but the mechanisms by which they are brought about are not understood. PCBs as well as PCB-Hs show low affinities for both alpha and beta estrogen receptor isoforms. In the present study we demonstrate that various environmentally relevant PCB-OHs are extremely potent inhibitors of human estrogen sulfotransferase, strongly suggesting that they indirectly induce estrogenic activity by increasing estradiol bioavailability in target tissues.

90%) by the haloDIMs at concentrations of 5 or 10 microM, and only 4, 4'-dichloroDIM alone increased cell proliferation. With the exception of 5,5'-difluoroDIM, the remaining compounds also inhibited E2-induced growth of MCF-7 human breast cancer cells. DihaloDIMs (100 mg/kg/dayx3) were not estrogenic in the immature female B6C3F1 mouse uterus; however, in animals co-treated with E2 (0.02 microg/mouse), 5,5'-dichloro- and 6,6'-dichloroDIM inhibited uterine progesterone receptor (PR) binding and uterine peroxidase activity, whereas 5,5'-dichloro- and 5,5'-dichloro-2,2'-dimethylDIM inhibited only the latter response. The antitumorigenic activities of the dihaloDIMs were determined by their inhibition of carcinogen-induced mammary tumor growth in female Sprague-Dawley rats. 4,4'-Dichloro-, 5,5'-dibromo- and 6,6'-dichloroDIM, significantly inhibited mammary tumor growth at doses of 1 mg/kg every second day, and no significant changes in organ weights or liver and kidney histopathology were observed. These three compounds were more active than DIM in the same in vivo assay.

Insulin-like growth factor-I (IGF-I), transforming growth factor alpha (TGFalpha) and epidermal growth factor (EGF) induced cathepsin D gene expression and reporter gene activity in MCF-7 human breast cancer cells transiently transfected with a construct (pCD1) containing a -2576 to -124 cathepsin D gene promoter insert. In contrast, IGF-I, but not TGFalpha or EGF, induced reporter gene activity in cells cotransfected with wild-type estrogen receptor (ER) expression plasmid and a construct (pCD2) containing estrogen-responsive downstream elements from -208 to -101. Promoter deletion and mutational analysis experiments identified four GC-rich sites and an imperfect palindromic estrogen responsive element required for IGF-I activation of the ER (ligand-independent). Subsequent studies with the mitogen-activated protein kinase (MAPK) inhibitor, PD98059, and a serine(118(-ER mutant confirmed the role of the MAPK pathway for IGF-I activation of the ER in MCF-7 cells. Thus, growth factor activation of ER can mediate transactivation vs ER/Sp1 binding to GC-rich sites and represents a novel pathway for ligand-independent ER action. The divergent pathways for IGF-I and TGFalpha/EGF activation of the ER observed in MCF-7 cells contrast with previous data indicating that pathways for growth factor activation of the ER are dependent on the gene and/or gene promoter and on cell context.

The uptake and subcellular partitioning of benzo[a]pyrene (BaP) were examined in a rat-liver cell line (Clone 9) using confocal and multiphoton microscopy. Following a 16-h treatment, intracellular accumulation of BaP increased with increasing concentration, and cytoplasmic BaP fluorescence reached saturation at 10 microM. Analysis of the kinetics of BaP uptake at this concentration indicated that BaP is rapidly partitioned into all cytoplasmic membranes within several min, although saturation was not reached until 4 h. Based upon the rapid uptake of BaP into membranes, the chronology of changes in gap junction-mediated intercellular communication (GJIC), plasma membrane potential (PMP), and steady state levels of intracellular Ca2+ in relation to the time-course for induction of microsomal ethoxyresorufin-0-deethylase (EROD) activity were examined. EROD activity in Clone 9 cells treated for 16 h increased with increasing concentrations of BaP and reached the highest levels at 40 microM BaP. In addition, kinetic analysis of EROD activity in Clone 9 cells treated with 10 microM BaP indicated that significant induction of EROD activity was not detected before 3 h, and it reached maximal levels by 16 h of treatment at this concentration. Both GJIC and PMP were directly affected by the partitioning of BaP into cellular membranes. The most sensitive index of BaP-induced changes in membrane function was GJIC which revealed a 25% suppression in cells exposed to 0.4 microM BaP for 16 h. Kinetic analysis revealed that suppression of GJIC occurred within 15 min of exposure of cells to 10 microM BaP, whereas significant suppression of PMP was not detected prior to 30-min exposure at this concentration. Elevation of basal Ca2+ level was also detected simultaneously with PMP at this dose. These data suggest that early changes in cellular membrane functions occur prior to detectable induction of EROD activity, although basal metabolic activation of BaP may contribute to these changes.

3,3',4,4'-Tetrachlorobiphenyl (tetraCB) binds to the aryl hydrocarbon receptor (AhR), and several reports have demonstrated that AhR agonists exhibit antiestrogenic and antitumorigenic activities in human breast cancer cells, the rodent uterus and breast. In contrast, a recent study showed that 3,3',4,4'-tetraCB bound the estrogen receptor (ER) and exhibited ER agonist activities, and we therefore have reinvestigated the estrogenic and antiestrogenic activities of 3,3',4,4'-tetraCB. Our results showed that 3,3',4,4'tetraCB and a structurally related analog, 3,3',4,4',5-pentaCB, did not bind the mouse uterine or human ER, did not induce proliferation of MCF-7 or T47D human breast cancer cells or induce reporter gene activity in cells transfected with E2-responsive constructs derived from the creatine kinase B (pCKB) or cathepsin D (pCD) gene promoters. Moreover, 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB did not induce an increase in uterine wet weight, peroxidase activity or progesterone receptor binding in the 21-25-day-old female B6C3F1 mouse uterus. In contrast, both compounds inhibited 17beta-estradiol (E2)-induced cell proliferation and transactivation in MCF-7/T47D cells and uterine responses in B6C3F1 mice; surprisingly inhibition of E2-induced reporter gene activity was not observed in T47D cells transfected with pCKB, and this was observed as a cell-specific response with other AhR agonists. Additionally, 3,3',4,4'-tetraCB significantly inhibited mammary tumor growth in female Sprague-Dawley rats initiated with 7,12-dimethylbenzanthracene. Our results indicate that 3,3',4,4'-tetraCB does not exhibit ER agonist activity but exhibits a broad spectrum of antiestrogenic responses consistent with ligand-mediated AhR-ER crosstalk.

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

There is considerable public, regulatory, and scientific concern regarding human exposure to endocrine-disrupting chemicals, which include compounds that directly modulate steroid hormone receptor pathways (estrogens, antiestrogens, androgens, antiandrogens) and aryl hydrocarbon receptor (AhR) agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Based on quantitative structure-activity relationships for both AhR and estrogen receptor (ER) agonists, the relative potency (RP) of individual compounds relative to a standard (e.g. TCDD and 17-beta-estradiol) have been determined for several receptor-mediated responses. Therefore, the TCDD or estrogenic equivalent (TEQ or EQ, respectively) of a mixture is defined as TEQ = sigma[T(i)]xRP(i)or EQ=sigma[E(i)]xRP(i), where T(i) and E(i) are concentrations of individual AhR or ER agonists in any mixture. This approach for risk assessment of endocrine-disrupting mixtures assumes that for each endocrine response pathway, the effects of individual compounds are essentially additive. This paper will critically examine the utility of the TEQ/EQ approach for risk assessment, the validity of the assumptions used for this approach, and the problems associated with comparing low dose exposures to xeno and natural (dietary) endocrine disruptors.

Acenaphthylene is a tricyclic aromatic hydrocarbon which induces hepatic methoxyresorufin O-demethylase (MROD) activity and CYP1A2 mRNA levels in 2 week-old male B6C3F1 mice. In the present study, this induction response was further investigated in genetically-inbred mice which differ in their aryl-hydrocarbon (Ah)-responsiveness. Acenapthylene (300 mg/kg) induced a 5- to 23-fold induction of MROD activity in Ah-nonresponsive (DBA and SJL) and responsive (C3H, C57/BL6, A/J, CBA and B6C3F1) mice. The highest induction response was observed in the DBA strain in which there was a 23- and 15-fold increase in activity in males and females, respectively. Acenaphthylene also caused a 2-fold increase in CYP1A2 mRNA and immunoreactive protein levels in 2 week-old DBA mice; however, this induction response was not observed in 6 week-old animals. For example, MROD activity in 6 week-old DBA mice was induced <2-fold by acenaphthylene, mainly as a consequence of increased basal CYP1A2 expression and MROD activity which, at the age of 6 weeks, approached levels induced by acenaphthylene in the 2 week-old mice. This was also observed by immunohistochemical staining with CYP1A2 antibodies of 2 and 6 week-old hepatic tissue from treated and control mice which also showed that CYP1A2 induction was dependent on the age of the animals.

Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.

Polychlorinated dibenzo-p-dioxins (PCDD), dibenzofurans (PCDF), and biphenyls (PCB) are industrial compounds or by-products that have been widely identified as environmental contaminants, and residues have been detected in fish, wildlife, and humans. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is the most toxic member of this class of halogenated aromatic hydrocarbons (HAH); mechanistic studies indicate that the toxic and biochemical effects associated with exposure to TCDD are mediated via initial binding to the cytosolic aryl hydrocarbon (Ah) receptor protein present in target tissues and organs. Several other 2,3,7, 8-substituted PCDD and PCDF and non-ortho substituted PCB also bind to the Ah receptor and induce toxic responses similar to those for TCDD. Moreover, for these HAH there is a rank order correlation between their structure-Ah receptor binding and structure-toxicity relationships, and this supports a role for the Ah receptor in mediating these responses. Thus, the toxic equivalency factor (TEF) approach for HAH is based on the common mechanism of action for TCDD and related compounds in which a TEF value for a "dioxin-like" congener is defined as the potency of the individual (i) congener relative to TCDD ([EC50 [TCDD]/EC50 [test compound]). The toxic or dioxin equivalent (TEQ) for a mixture of HAH is defined by the following equation: TEQ = sigma [PCDDi] x TEFi + sigma [PCDFi] x TEFi. Industrial emissions and environmental and food residues contain complex mixtures of HAH (exodioxins) and the TEF/TEQ approach is used to regulate emissions and estimate the potential exposure and possible adverse health effects of exodioxins. The TEF approach for risk assessment of exodioxins makes a number of assumptions, including response additivity for individual compounds in a mixture of HAH. This review documents some of the following problems and limitations of the TEF approach: 1) environmental and food residues of HAH contain "non-dioxin-like" PCB that exhibit "antidioxin" activity for some responses; 2) the human diet contains endogenous Ah receptor ligands (endodioxins) such as polynuclear aromatic hydrocarbons (PAH), aromatic amines in cooked foods, indole-3-carbinol (I3C), and related hetero-PAH in cruciferous vegetables. Mass balance and mass potency estimates for human dietary intakes suggest that for some responses the effects of natural or endodioxins may be greater than those of exodioxins; and 3) I3C, a weak Ah receptor agonist, also exhibits Ah receptor antagonist activity, and interactions between I3C and endodioxins may inhibit or inactivate some toxic responses and decrease TEQ(Exodioxin).

Halogenated aromatic hydrocarbons (HAHs), such as polychlorinated biphenyls (PCBs) and dibenzo-p-dioxins (PCDDs), alter cognitive function and learning. The cellular basis of HAH-induced alteration of brain function is not well-understood. The hippocampus is a likely site of toxic action because of its well-known roles in learning and memory, as well as its propensity to accumulate environmental neurotoxicants. A hippocampal function that can be measured readily is evoked excitatory postsynaptic potentials (EPSPs), which are an index of excitatory synaptic function. In this study, effects of HAHs on EPSPs were characterized in hippocampal slices from adolescent to adult male Sprague-Dawley rats. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4-TCDD were used because these HAHs are prototypical potent and weak aryl hydrocarbon (Ah) receptor agonists, respectively. 2,2',5,5'-Tetrachlorobiphenyl (TCB) was used as a prototypical ortho-substituted PCB, which acts through Ah receptor-independent pathways. For each hippocampal slice, peak amplitudes of EPSPs during a 15-min recording period (1 recording/min) were averaged and used as baseline (100%). Subsequent EPSPs were expressed as percentage of baseline. TCDD and 1,2,3,4-TCDD did not alter EPSPs in slices from the middle third of the hippocampus. However, in ventral slices, TCDD significantly decreased EPSPs, whereas 1,2,3,4-TCDD was inactive. TCB decreased EPSPs in both middle and ventral slices at half-maximal stimulation. An unexpected reversal of inhibition was observed within 30 min of continuous application of TCDD or TCB. In ventral slices, L-type calcium channel blocker nifedipine blocked inhibition of EPSPs induced by TCDD but not EPSPs inhibited by TCB. These results suggest that, while TCB-induced inhibition of EPSPs occurs through an unknown mechanism, TCDD-induced inhibition of EPSPs was mediated by L-type calcium channel activity in a congener-specific manner.

Development of breast cancer in women is dependent on diverse factors, including genetic predisposition, exposure to both exogenous and endogenous chemicals, which can modulate initiation, promotion and progression of this disease, and the timing of exposure to these agents. Several compounds--including 16 alpha-hydroxyestrone (16 alpha-OHE1), catecholestrogens, and aromatic amines--have been proposed as initiators of mammary carcinogenesis in humans; however, their role as genotoxins is unconfirmed. Lifetime exposure to estrogens has been established as an important risk factor for breast cancer, and it has been suggested that xenoestrogens may directly add to the hormonal risk or indirectly increase risk by decreasing 2-hydroxyestrone (2-OHE1)/16 alpha-OHE1 metabolite ratios. Results of recent studies suggest that chemical-induced modulation of 2-OHE1/16 alpha-OHE1 metabolite ratios is not predictive of xenoestrogens or mammary carcinogens. Moreover, based on current known dietary intakes of natural and xenoestrogenic/antiestrogenic chemicals, it is unlikely that xenoestrogens contribute significantly to a woman's overall lifetime exposure to estrogens. More information is required on the identities and serum levels of both natural and xenoendocrine active compounds, their concentrations in serum, and the mammary gland and levels of these compounds at critical periods of exposure.

T47D human breast cancer cells were cultured in estrogen-deficient media for up to 32 months and the resulting cell line (L(hE(-))) exhibited unique phenotypic and genotypic characteristics. Compared to low passage (L) cells, the L(hE(-)) cells exhibited a significantly higher rate of proliferation, unique morphological features, advanced ploidy status and 5- to 10-fold higher levels of the estrogen receptor (ER) as determined by ligand binding and Western blot analysis. Sequence analysis of the DNA binding domain of the ER revealed a C-->A transversion which resulted in a H513N amino acid change. Treatment of L cells with 10 n m 17beta-estradiol (E2) resulted in a greater than two-fold increase in cell proliferation which was inhibited by tamoxifen, 4'-hydroxytamoxifen, ICI 164,384 and ICI 182,780. In contrast, 10 n m E2 caused a 70% decrease in growth of L(hE(-)) cells and this antimitogenic activity was blocked by ICI 164,384 and ICI 182,780 but not by tamoxifen or 4'-hydroxytamoxifen. L(hE(-)) cells were E2-responsive in transient transfection studies using a plasmid containing an estrogen-responsive element derived from the vitellogenin A2 gene promoter. These data show that the phenotypic and genotypic characteristics of L(hE(-)) T47D cells resemble those described for ER-negative cell lines stably transfected with the ER., (Copyright 1998 Academic Press.)

Several studies have reported that polychlorinated biphenyls (PCBs) exhibit estrogenic activity; however, it is not clear if these responses are associated with the polychlorinated hydrocarbon or its hydroxylated metabolite. In order to further test this hypothesis, a battery of in vitro and in vivo assays were used to investigate the estrogenic and antiestrogenic activities of 2,4,6,2',6'-pentachlorobiphenyl (PCB 104), its para-hydroxylated derivative 2,4,6,2',6'-pentachloro-4-biphenylol (HO-PCB 104), and its para-chlorinated derivative 2,4,6,2',4',6'-hexachlorobiphenyl (PCB 155). PCB 104 was found to 1) compete with tritiated 17beta-estradiol (E2) for binding to the mouse uterine estrogen receptor (ER); 2) induce gene expression in MCF-7 human breast cancer cells transiently transfected with the Gal4-human ER chimeric construct (Gal4-HEGO) and the Gal4-regulated luciferase reporter gene (17m5-G-Luc); and 3) increase MCF-7 cell proliferation in a dose-dependent manner. HO-PCB 104 exhibited greater estrogenic activity than PCB 104 in the in vitro assays examined. However, gas chromatographic-mass spectrophotometric analysis of extracts prepared from MCF-7 cells incubated with PCB 104 failed to detect the presence of the expected major metabolite HO-PCB 104. The estrogenic activity of the para-chlorinated derivative, PCB 155, was minimal compared to PCB 104 and HO-PCB 104, but it did exhibit significant antiestrogenic activity following co-treatment with 1 nM E2. Co-treatment of PCB 104 with 1 nM E2 had no effect on reporter gene expression compared to E2 alone, while 10 microM HO-PCB 104 exhibited additivity with 1 nM E2. At a dose of 202 mg/kg,PCB 104 increased uterine wet weight in ovariectomized CD-1 mice and induced vaginal epithelial cell cornification at 202, 16, and 1.7 mg/kg in a dose-dependent manner. These studies demonstrate that in addition to the hydroxylated metabolites, selected parent PCB congeners may also exhibit estrogenic and antiestrogenic activities.