Epigallocatechin-3-gallate does not affect the activity of enzymes involved in metabolic activation and cellular excretion of benzo[a]pyrene in human colon carcinoma cells.

Benzo[a]pyrene (B[a]P) and related procarcinogens found in cigarette smoke and roasted foodstuff require metabolic activation to build mutagenic DNA adducts that may cause tumor diseases like colorectal cancer. The major B[a]P-activating enzymes belong to the cytochrome-P450 (CYP)-1 family and are regulated by the aryl hydrocarbon receptor (AhR). Previous studies have indicated that an inhibition of AhR is accompanied with a reduced metabolic activation of B[a]P and therefore may act protective against carcinogenesis. We investigated if the green tea flavonoid (-)-epigallocatechin-3-gallate (EGCG), a known AhR inhibitor, is able to influence B[a]P-metabolizing and B[a]P-transporting enzymes in human Caco-2 colon carcinoma cells. Strikingly, treatment with EGCG did neither affect constitutive and B[a]P-inducible expression of CYP1A1 and UDP-glucuronosyltransferase (UGT)-1A1 nor overall CYP1 and UGT enzyme activities, indicating that EGCG does not antagonize the AhR in Caco-2 cells. Since flavonoids were also identified to enhance the activity of B[a]P-carrying transporter, we analyzed if EGCG exposure alters cellular excretion of B[a]P conjugates. In contrast to the positive control fisetin, EGCG did not affect cellular excretion of B[a]P metabolites. Our data provide evidence that EGCG does not alter the metabolism and transport of B[a]P in Caco-2 cells, and thus may not protect against procarcinogenic food contaminants.

Species-specific differential AhR expression protects human neural progenitor cells against developmental neurotoxicity of PAHs.

BACKGROUND: Because of their lipophilicity, persistent organic pollutants (POPs) cross the human placenta, possibly affecting central nervous system development. Most POPs are known aryl hydrocarbon receptor (AhR) ligands and activators of AhR signaling. Therefore, AhR activation has been suggested to cause developmental neurotoxicity (DNT). OBJECTIVE: We studied the effects of AhR ligands on basic processes of brain development in two comparative in vitro systems to determine whether AhR-activation is the underlying mechanism for reported DNT of POPs in humans. METHODS: We employed neurosphere cultures based on human neural progenitor cells (hNPCs) and wild-type and AhR-deficient mouse NPCs (mNPCs) and studied the effects of different AhR agonists [3-methylcholanthrene (3-MC), benzo(a)pyrene [B(a)P], and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)] and an antagonist [3'-methoxy-4'-nitroflavone (MNF)] on neurosphere development. Moreover, we analyzed expression of AhR and genes involved in AhR signaling. RESULTS: In contrast to wild-type mNPCs, hNPCs and AhR-deficient mNPCs were insensitive to AhR agonism or antagonism. Although AhR modulation attenuated wild-type mNPC proliferation and migration, hNPCs and AhR-deficient mNPCs remained unaffected. Results also suggest that species-specific differences resulted from nonfunctional AhR signaling in hNPCs. CONCLUSION: Our findings suggest that in contrast to wild-type mNPCs, hNPCs were protected against polycyclic aromatic hydrocarbon-induced DNT because of an absence of AhR This difference may contribute to species-specific differences in sensitivity to POPs.

Regulation of dioxin receptor function by different beta-carboline alkaloids.

The dioxin receptor, also known as arylhydrocarbon receptor (AhR), is a ligand-activated transcription factor that mediates the toxicity of dioxins and related environmental contaminants. In addition, there is a growing list of natural compounds, mainly plant polyphenols that can modulate AhR function and downstream signaling with quite unknown consequences for cellular function. We investigate the potential of four different beta-carboline alkaloids to stimulate AhR signaling in human hepatoma cells and keratinocytes. Three test substances, namely rutaecarpine, annomontine and xestomanzamine A, increase AhR-driven reporter gene activity as well as expression of two AhR target genes in a dose-dependent and time-dependent manner. Additionally, the three test alkaloids stimulate cytochrome P450 (CYP) 1 enzyme activity without showing any antagonistic effects regarding benzo(a)pyrene-stimulated CYP1 activation. The AhR-activating property of the beta-carbolines is completely abrogated in AhR-deficient cells providing evidence that rutaecarpine, annomontine and xestomanzamine A are natural stimulators of the human AhR. The toxicological relevance of beta-carboline-mediated AhR activation is discussed.

Luteolin enhances the bioavailability of benzo(a)pyrene in human colon carcinoma cells.

We investigated the effect of luteolin, a plant-derived flavonoid, on benzo(a)pyrene (B(a)P)-stimulated drug metabolism and transport in human colon carcinoma cells. While luteolin treatment inhibited B(a)P-induced expression and activity of arylhydrocarbon receptor-dependent cytochrome P450 enzymes, the overall activity of UDP-glucuronosyltransferases and sulfotransferases was not affected by luteolin, indicating that luteolin affects phase-I but not phase-II function. Luteolin exposure decreased apical transport of B(a)P metabolites due to its interaction with the transporter breast cancer resistance protein. Inhibitor studies provide a first clue to the mechanism of luteolin-mediated inhibition of this transporter. The inhibition of both phase-I metabolism as well as phase-III transport by luteolin resulted in a 3-fold intracellular accumulation of radioactively labeled B(a)P. Our data reveal that luteolin is able to interfere with crucial steps of drug metabolism and thereby enhances the bioavailability of B(a)P. These findings are of special importance regarding future benefit-risk evaluations of preventive flavonoid usage.

Growth factors, cytokines and their receptors as downstream targets of arylhydrocarbon receptor (AhR) signaling pathways.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant, which causes a variety of severe health effects, e.g. immunosuppression, hepatotoxicity, and carcinogenesis. The main mediator of TCDD toxicity is the arylhydrocarbon receptor (AhR), which, upon activation, translocates into the nucleus and enforces gene expression. Since most of the pleiotropic effects caused by TCDD are associated with alterations in cell growth and differentiation, the analysis of the interference of the AhR with factors controlling these cellular functions seems to be a promising target regarding the prevention and treatment of chemical-provoked diseases. Cell growth and differentiation are regulated by numerous growth factors and cytokines. These multifunctional peptides promote or inhibit cell growth and regulate differentiation and other cellular processes, depending on cell-type and developmental stage. They are involved in the regulation of a broad range of physiological processes, including immune response, hematopoiesis, neurogenesis, and tissue remodeling. The complex network of growth factors and cytokines is accurately regulated and disturbances of this system are associated with adverse health effects. The molecular mechanisms by which the AhR interferes with this signaling network are multifaceted and the physiological consequences of this cross-talk are quite enigmatic. The investigation of this complex interaction is an exciting task, especially with respect to the recently described non-genomic and/or ligand-independent activities of AhR. Therefore, we summarize the current knowledge about the interaction of the AhR with three cytokine-/growth factor-related signal transducers -- the epidermal growth factor (EGF) family, tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta) -- with regard to pathophysiological findings.

Analysis of the transcriptional regulation and molecular function of the aryl hydrocarbon receptor repressor in human cell lines.

The aryl hydrocarbon receptor repressor (AhRR) is a member of the aryl hydrocarbon receptor (AhR) signaling cascade, which mediates dioxin toxicity and is involved in regulation of cell growth and differentiation. The AhRR was described as a feedback modulator, which counteracts AhR-dependent gene expression. We investigated the molecular mechanisms of transcriptional regulation of the human AhRR by cloning its regulatory DNA region located in intron I of the AhRR. By means of reporter gene analyses and generation of deletion variants, we identified a functional, 3-methylcholanthrene-sensitive xenobiotic response element (XRE) site. Chromatin immunoprecipitation analyses revealed that the AhRR binds to this XRE, displaying an autoregulatory loop of AhRR expression. In addition we show that an adjacent GC-box is of functional relevance for AhRR transcription, since blocking of this GC-box resulted in a decrease of constitutive and inducible AhRR gene activity. The differences in constitutive AhRR mRNA level observed in HepG2, primary fibroblast, and HeLa cells are directly correlated with CYP1A1 inducibility. We show that the nonresponsiveness of high AhRR-expressing cells toward AhR-agonists is associated with a constitutive binding of the AhRR to XRE sites of CYP1A1. Treatment with the histone deacetylase inhibitor sodium butyrate restored the responsiveness of CYP1A1 in these cell lines, due to the dissociation of AhRR from the XREs. Furthermore, transient AhRR mRNA silencing in untreated HeLa cells was accompanied by an increase of basal CYP1A1 expression, pointing to a constitutive role of the AhRR in regulation of CYP1A1. The functional relevance of the AhRR in high AhRR-expressing primary fibroblasts is discussed.