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Urban wildfires may generate numerous unidentified chemicals of toxicity concern. Ash samples were collected from burned residences and from an undeveloped upwind reference site, following the Tubbs fire in Sonoma County, California. The solvent extracts of ash samples were analyzed using GC- and LC-high-resolution mass spectrometry (HRMS) and using a suite of in vitro bioassays for their bioactivity toward nuclear receptors [aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and androgen receptor (AR)], their influence on the expression of genetic markers of stress and inflammation [interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2)], and xenobiotic metabolism [cytochrome P4501A1 (CYP1A1)]. Genetic markers (CYP1A1, IL-8, and COX-2) and AhR activity were significantly higher with wildfire samples than in solvent controls, whereas AR and ER activities generally were unaffected or reduced. The bioassay responses of samples from residential areas were not significantly different from the samples from the reference site despite differing chemical compositions. Suspect and nontarget screening was conducted to identify the chemicals responsible for elevated bioactivity using the multiple streams of HRMS data and open-source data analysis workflows. For the bioassay endpoint with the largest available database of pure compound results (AhR), nontarget features statistically related to whole sample bioassay response using Spearman's rank-order correlation coefficients or elastic net regression were significantly more likely (by 10 and 15 times, respectively) to be known AhR agonists than the overall population of compounds tentatively identified by nontarget analysis. The findings suggest that a combination of nontarget analysis, in vitro bioassays, and statistical analysis can identify bioactive compounds in complex mixtures.

Diverse organic compounds, many derived from consumer products, are found in sewage sludge worldwide. Understanding which of these poses the most significant environmental threat following land application can be investigated through a variety of predictive and cell-based toxicological techniques. Nontargeted analysis using high-resolution mass spectrometry with predictive estrogenic activity modeling was performed on sewage sludge samples from 12 wastewater treatment plants in California. Diisobutyl phthalate and dextrorphan were predicted to exhibit estrogenic activity and identified in >75% of sludge samples, signifying their universal presence and persistence. Additionally, the application of an estrogen-responsive cell bioassay revealed reductions in agonistic activity during mesophilic and thermophilic treatment but significant increases in antagonism during thermophilic treatment, which warrants further research. Ten nontarget features were identified (metoprolol, fenofibric acid, erythrohydrobupropion, oleic acid, mestranol, 4'-chlorobiphenyl-2,3-diol, medrysone, scillarenin, sudan I, and N,O-didesmethyltramadol) in treatment set samples and are considered to have influenced the in vitro estrogenic activity observed. The combination of predictive and in vitro estrogenicity with nontargeted analysis has led to confirmation of 12 estrogen-active contaminants in California sewage sludge and has highlighted the importance of evaluating both agonistic and antagonistic responses when evaluating the bioactivity of complex samples.

Air pollution poses a serious risk to human health. To help understand the contribution of smoke from wood burning to the harmfulness of air pollution toward the skin, we studied the effects of liquid smoke, aqueous extracts of wood smoke condensate, a commercially available food flavor additive, in cultured keratinocytes. We report that liquid smoke can react with and cross-link keratinocyte cellular proteins, leading to abnormal cross-linked envelope formation. Instead of inducing genes ordinarily involved in terminal differentiation, liquid smoke induced expression of genes associated with stress responses. When transglutaminase activity was inhibited, liquid smoke still promoted protein cross-linking and envelope formation in keratinocytes. This phenomenon likely results from oxidative stress and protein adducts from aldehydes as either preloading the cells with N-acetylcysteine or reducing the aldehyde content of liquid smoke decreased its ability to promote protein cross-linking and envelope formation. Finally, liquid smoke-induced envelopes were found to have elevated protein content, suggesting oxidative cross-linking and formation of protein adducts might impair barrier function by inducing abnormal incorporation of cellular proteins into envelopes. Since the cross-linked protein envelope provides structural stability to the stratum corneum and serves as a scaffold for the organization of the corneocyte lipid envelope (hydrophobic barrier to the environment), these findings provide new insight into the mechanism by which pro-oxidative air pollutants can impair epidermal function.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of an AhR lacking the entire PASB structurally diverse chemicals, including halogenated aromatic hydrocarbons. Ligand-dependent transformation of the AhR into its DNA binding form involves a ligand-dependent conformational change, heat shock protein 90 (hsp90), dissociation from the AhR complex and AhR dimerization with the AhR nuclear translocator (ARNT) protein. The mechanism of AhR transformation was examined using mutational approaches and stabilization of the AhR:hsp90 complex with sodium molybdate. Insertion of a single mutation (F281A) in the hsp90-binding region of the AhR resulted in its constitutive (ligand-independent) transformation/DNA binding in vitro. Mutations of AhR residues within the Arg-Cys-rich region (R212A, R217A, R219A) and Asp371 (D371A) impaired AhR transformation without a significant effect on ligand binding. Stabilization of AhR:hsp90 binding with sodium molybdate decreased transformation/DNA binding of the wild type AhR but had no effect on constitutively active AhR mutants. Interestingly, transformation of the AhR in the presence of molybdate allowed detection of an intermediate transformation ternary complex containing hsp90, AhR, and ARNT. These results are consistent with a stepwise transformation mechanism in which binding of ARNT to the liganded AhR:hsp90 complex results in a progressive displacement of hsp90 and conversion of the AhR into its high affinity DNA binding form. The available molecular insights into the signaling mechanism of other Per-ARNT-Sim (PAS) domains and structural information on hsp90 association with other client proteins are consistent with the proposed transformation mechanism of the AhR.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of an AhR lacking the entire PASB structurally diverse chemicals, including halogenated aromatic hydrocarbons. Ligand-dependent transformation of the AhR into its DNA binding form involves a ligand-dependent conformational change, heat shock protein 90 (hsp90), dissociation from the AhR complex and AhR dimerization with the AhR nuclear translocator (ARNT) protein. The mechanism of AhR transformation was examined using mutational approaches and stabilization of the AhR:hsp90 complex with sodium molybdate. Insertion of a single mutation (F281A) in the hsp90-binding region of the AhR resulted in its constitutive (ligand-independent) transformation/DNA binding in vitro. Mutations of AhR residues within the Arg-Cys-rich region (R212A, R217A, R219A) and Asp371 (D371A) impaired AhR transformation without a significant effect on ligand binding. Stabilization of AhR:hsp90 binding with sodium molybdate decreased transformation/DNA binding of the wild type AhR but had no effect on constitutively active AhR mutants. Interestingly, transformation of the AhR in the presence of molybdate allowed detection of an intermediate transformation ternary complex containing hsp90, AhR, and ARNT. These results are consistent with a stepwise transformation mechanism in which binding of ARNT to the liganded AhR:hsp90 complex results in a progressive displacement of hsp90 and conversion of the AhR into its high affinity DNA binding form. The available molecular insights into the signaling mechanism of other Per-ARNT-Sim (PAS) domains and structural information on hsp90 association with other client proteins are consistent with the proposed transformation mechanism of the AhR.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of an AhR lacking the entire PASB structurally diverse chemicals, including halogenated aromatic hydrocarbons. Ligand-dependent transformation of the AhR into its DNA binding form involves a ligand-dependent conformational change, heat shock protein 90 (hsp90), dissociation from the AhR complex and AhR dimerization with the AhR nuclear translocator (ARNT) protein. The mechanism of AhR transformation was examined using mutational approaches and stabilization of the AhR:hsp90 complex with sodium molybdate. Insertion of a single mutation (F281A) in the hsp90-binding region of the AhR resulted in its constitutive (ligand-independent) transformation/DNA binding in vitro. Mutations of AhR residues within the Arg-Cys-rich region (R212A, R217A, R219A) and Asp371 (D371A) impaired AhR transformation without a significant effect on ligand binding. Stabilization of AhR:hsp90 binding with sodium molybdate decreased transformation/DNA binding of the wild type AhR but had no effect on constitutively active AhR mutants. Interestingly, transformation of the AhR in the presence of molybdate allowed detection of an intermediate transformation ternary complex containing hsp90, AhR, and ARNT. These results are consistent with a stepwise transformation mechanism in which binding of ARNT to the liganded AhR:hsp90 complex results in a progressive displacement of hsp90 and conversion of the AhR into its high affinity DNA binding form. The available molecular insights into the signaling mechanism of other Per-ARNT-Sim (PAS) domains and structural information on hsp90 association with other client proteins are consistent with the proposed transformation mechanism of the AhR.

Ligand-dependent activation of the Ah receptor (AhR) can result in an extremely diverse spectrum of biological and toxic effects that occur in a ligand-, species- and tissue-specific manner. While the classical mechanism of AhR-dependent signal transduction is directly related to its ability to modulate gene expression, the dramatic diversity in responses observed following AhR activation or inhibition is inconsistent with a single molecular mechanism of AhR action. Recent studies have revealed that key molecular events underlying the AhR signaling pathway are significantly more varied and complex than previously established, and the specificity and diversity in AhR response can be selectively modulated by a variety of factors. Here we describe new insights into the mechanistic diversity in AhR signal transduction that can contribute to ligand-, species- and tissue-specific differences in AhR reponse.

Ligand-dependent activation of the Ah receptor (AhR) can result in an extremely diverse spectrum of biological and toxic effects that occur in a ligand-, species- and tissue-specific manner. While the classical mechanism of AhR-dependent signal transduction is directly related to its ability to modulate gene expression, the dramatic diversity in responses observed following AhR activation or inhibition is inconsistent with a single molecular mechanism of AhR action. Recent studies have revealed that key molecular events underlying the AhR signaling pathway are significantly more varied and complex than previously established, and the specificity and diversity in AhR response can be selectively modulated by a variety of factors. Here we describe new insights into the mechanistic diversity in AhR signal transduction that can contribute to ligand-, species- and tissue-specific differences in AhR reponse.

Organic aerosols generated from the smoldering combustion of wood critically impact air quality and health for billions of people worldwide; yet, the links between the chemical components and the optical or biological effects of woodsmoke aerosols (WSA) are still poorly understood. In this work, an untargeted analysis of the molecular composition of smoldering WSA, generated in a controlled environment from nine types of heartwood fuels (African Mahogany, Birch, Cherry, Maple, Pine, Poplar, Red Oak, Redwood, and Walnut) identified several hundred compounds using gas chromatography mass spectrometry (GC-MS) and nano-electrospray high-resolution mass spectrometry (HRMS) with tandem multistage mass spectrometry (MSn). The effects of WSA on cell toxicity, aryl hydrocarbon receptor (AhR)- and estrogen receptor (ER)-dependent gene expression were characterized with cellular assays, and the visible mass absorption coefficients (MACvis) of WSA were measured with UV-visible spectroscopy. The WSA studied in this work have significant levels of biological and toxicological activity, with exposure levels in both an outdoor and indoor environment similar to or greater than those of other toxicants. A correlation between the HRMS molecular composition and aerosol properties found that phenolic compounds from the oxidative decomposition of lignin were the main drivers of aerosol effects, while the cellulose decomposition products played a secondary role, e.g., levoglucosan was anti-correlated with multiple effects. Polycyclic aromatic hydrocarbons (PAHs) were not expected to form at the combustion temperature in this work, nor were observed above the detection limit; thus, biological and optical properties of the smoldering WSA are not attributed to PAHs. Syringyl compounds tend to correlate with cell toxicity, while the more conjugated molecules (including several compounds assigned to dimers) had higher AhR activity and MACvis. The negative correlation between cell tox

Organic aerosols generated from the smoldering combustion of wood critically impact air quality and health for billions of people worldwide; yet, the links between the chemical components and the optical or biological effects of woodsmoke aerosols (WSA) are still poorly understood. In this work, an untargeted analysis of the molecular composition of smoldering WSA, generated in a controlled environment from nine types of heartwood fuels (African Mahogany, Birch, Cherry, Maple, Pine, Poplar, Red Oak, Redwood, and Walnut) identified several hundred compounds using gas chromatography mass spectrometry (GC-MS) and nano-electrospray high-resolution mass spectrometry (HRMS) with tandem multistage mass spectrometry (MSn). The effects of WSA on cell toxicity, aryl hydrocarbon receptor (AhR)- and estrogen receptor (ER)-dependent gene expression were characterized with cellular assays, and the visible mass absorption coefficients (MACvis) of WSA were measured with UV-visible spectroscopy. The WSA studied in this work have significant levels of biological and toxicological activity, with exposure levels in both an outdoor and indoor environment similar to or greater than those of other toxicants. A correlation between the HRMS molecular composition and aerosol properties found that phenolic compounds from the oxidative decomposition of lignin were the main drivers of aerosol effects, while the cellulose decomposition products played a secondary role, e.g., levoglucosan was anti-correlated with multiple effects. Polycyclic aromatic hydrocarbons (PAHs) were not expected to form at the combustion temperature in this work, nor were observed above the detection limit; thus, biological and optical properties of the smoldering WSA are not attributed to PAHs. Syringyl compounds tend to correlate with cell toxicity, while the more conjugated molecules (including several compounds assigned to dimers) had higher AhR activity and MACvis. The negative correlation between cell tox

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [³H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma. They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit. The master regulators driving the expression of resistance-genes remain poorly understood. Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes. Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse. Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists. We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation. Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth. We thus propose AhR-impairment as a strategy to overcome melanoma resistance.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [³H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma. They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit. The master regulators driving the expression of resistance-genes remain poorly understood. Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes. Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse. Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists. We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation. Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth. We thus propose AhR-impairment as a strategy to overcome melanoma resistance.

In the present study 42 polycyclic aromatic compounds (PACs) were investigated for their estrogenic potential using the VM7Luc4E2 transactivation assay. Relative potencies were determined for mass-balance analysis. In addition, compounds were tested in combination with the estrogen receptor (ER) antagonist vertical bar C vertical bar 182,780 (vertical bar C vertical bar) and the aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor a-naphthoflavone. Luciferase induction and CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity were measured to assess whether the estrogenic activity was elicited by the compound itself and/or by its metabolites. Relative potencies ranged between 10(-7) and 10(-4). The ability of ICI to decrease luciferase activity stimulated by all compounds indicated that the induction responses were ER-dependent. The aryl hydrocarbon receptor antagonist/CYP1A1 inhibitor a-naphthoflavone decreased luciferase induction and EROD activity by several compounds, including the methylated chrysenes, suggesting that metabolites of these chemicals contributed to ER activation. Several PACs, such as acridine and its derivatives, appear to directly activate the ER. Furthermore, extracts of soils from industrial areas were examined using this bioassay, and estrogenic activity was detected in all soil samples. Mass-balance analysis using a combination of relative potencies and chemical analysis of the samples suggested that polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs, such as 1-and 3-methylchrysene, are important contributors to the overall estrogenic activity. However, these results revealed that a considerable proportion of the estrogenic activity in the soil remained unexplained, indicating the presence of other significant estrogenic compounds.

Laboratory safety requires protecting personnel from chemical exposures. Working with stock solutions of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDFs) in routine analysis of feed and food with bioanalytical or physicochemical methods raises some concerns. Since PCDD/PCDFs are considered as possibly acutely toxic, the potential risks were evaluated to determine whether supervision of their use is necessary. Based on LD50-data for oral or dermal intake, hazard classification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a substance (category 1) and in commercially available TCDD standard solutions (category 4) is different. As worst case exposure scenario during routine laboratory work it was assumed that a dose of 100 ng TCDD gets onto the skin and is absorbed. This would result in the total body burden of a 70 kg person with 15 kg fat increasing from 10 (upper range of current background levels) to ∼17 pg of toxic equivalents (TEQs) of PCDD/PCDFs per g lipid, a level commonly observed over past decades. Chloracne, the main acute effect occurring weeks after exposure, is observed at much higher blood concentrations than estimated from accidental laboratory exposure. Immunotoxicity, developmental effects and other toxic effects may occur at lower blood levels, but require longer periods to develop. Since acute toxic symptoms don't occur within an “8 h acute time window”, no supervision is necessary when working with standard solutions in routine analysis. Nevertheless, precautionary measures are needed regarding long-term adverse health effects and appropriate workplace conditions must exist to ensure that additional occupational exposure to PCDD/PCDFs by laboratory personnel is negligible.

1,4-Dihydroxy-2-naphthoic acid (1,4-DHNA) is a bacterial-derived metabolite that binds the aryl hydrocarbon receptor (AhR) and exhibits anti-inflammatory activity in the gut. The structure-dependent AhR activity of hydroxyl/carboxy-substituted naphthoic acids (NAs) was determined in young adult mouse colonic (YAMC) cells and human Caco2 colon cancer cells using CYP1A1/CYP1B1 mRNAs as Ah-responsive genes. Compounds used in this study include 1,4-, 3,5-, and 3,7-DHNA, 1,4-dimethoxy-2-naphthoic acid (1,4-DMNA), 1- and 4-hydroxy-2-naphthoic acid (1-HNA, 4-HNA), 1- and 2-naphthoic acid (1-NA, 2-NA), and 1- and 2-naphthol (1-NOH, 2-NOH). 1,4-DHNA was the most potent compound among hydroxyl/carboxy naphthalene derivatives, and the fold induction response for CYP1A1 and CYP1B1 was similar to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in YAMC and Caco2 cells. 1- and 4-HNA were less potent than 1,4-DHNA but induced maximal (TCDD-like) response for CYP1B1 (both cell lines) and CYP1A1 (Caco2 cells). With the exception of 1- and 2-NA, all compounds significantly induced Cyp1b1 in YAMC cells and these responses were not observed in AhR-deficient YAMC cells generated using CRISPR/Cas9 technology. In addition, we also observed that 1- and 2-NOH (and 1,4-DHNA) were weak AhR agonists, and 1- and 2-NOH also exhibited partial AhR antagonist activity. Structure-activity relationship studies for CYP1A1 but not CYP1B1 were similar in both cell lines, and CYP1A1 induction required one or both 1,4-dihydroxy substituents and activity was significantly enhanced by the 2-carboxyl group. We also used computational analysis to show that 1,4-DHNA and TCDD share similar interactions within the AhR binding pocket and differ primarily due to the negatively charged group of 1,4-DHNA.

The toxic effects of dioxins and related compounds (DRCs) are mediated by the aryl hydrocarbon receptor (AHR). Our previous study identified AHR1 and AHR2 genes from the red seabream (Pagrus major). Moreover, we found that AHR2 mRNA levels were notably elevated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in the early life stage of red seabream embryos, while AHR1 mRNA level was not altered. In this study, to investigate the regulatory mechanism of these AHR transcripts, we cloned and characterized 5'-flanking regions of AHR1 and AHR2 genes. Both of the 5'-flanking regions in these AHR genes contained three potential xenobiotic-responsive elements (XREs). To assess whether the 5'-flanking region is transactivated by rsAHR1 and rsAHR2 proteins, we measured the transactivation potency of the luciferase reporter plasmids containing the 5'-flanking regions by AHR1 and AHR2 proteins that were transiently co-expressed in COS-7. Only reporter plasmid (pGL4-rsAHR2-3XREs) that contained three putative XRE sites in the 5'-flanking region of AHR2 gene showed a clear TCDD dose-dependent transactivation by AHR1 and AHR2 proteins. TCDD-EC50 values for the rsAHR2-derived XRE transactivation were 1.3 and 1.4 nM for AHR1 and AHR2, respectively. These results suggest that the putative XREs of AHR2 gene have a function for AHR1- and AHR2-mediated transactivation, supporting our in ovo observation of an induction of AHR2 mRNA levels by TCDD exposure. Mutations in XREs of AHR2 gene led to a decrease in luciferase induction. Electrophoretic mobility shift assay showed that XRE1, the closest XRE from the start codon in AHR2 gene, is mainly responsible for the binding with TCDD-activated AHR. This suggests that TCDD-activated AHR1 and AHR2 up-regulate the AHR2 mRNA levels and this auto-induced AHR2 may amplify the signal transduction of its downstream targets including CYP1A in the red seabream.

The toxic effects of dioxins and related compounds (DRCs) are mediated by the aryl hydrocarbon receptor (AHR). Our previous study identified AHR1 and AHR2 genes from the red seabream (Pagrus major). Moreover, we found that AHR2 mRNA levels were notably elevated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in the early life stage of red seabream embryos, while AHR1 mRNA level was not altered. In this study, to investigate the regulatory mechanism of these AHR transcripts, we cloned and characterized 5'-flanking regions of AHR1 and AHR2 genes. Both of the 5'-flanking regions in these AHR genes contained three potential xenobiotic-responsive elements (XREs). To assess whether the 5'-flanking region is transactivated by rsAHR1 and rsAHR2 proteins, we measured the transactivation potency of the luciferase reporter plasmids containing the 5'-flanking regions by AHR1 and AHR2 proteins that were transiently co-expressed in COS-7. Only reporter plasmid (pGL4-rsAHR2-3XREs) that contained three putative XRE sites in the 5'-flanking region of AHR2 gene showed a clear TCDD dose-dependent transactivation by AHR1 and AHR2 proteins. TCDD-EC50 values for the rsAHR2-derived XRE transactivation were 1.3 and 1.4 nM for AHR1 and AHR2, respectively. These results suggest that the putative XREs of AHR2 gene have a function for AHR1- and AHR2-mediated transactivation, supporting our in ovo observation of an induction of AHR2 mRNA levels by TCDD exposure. Mutations in XREs of AHR2 gene led to a decrease in luciferase induction. Electrophoretic mobility shift assay showed that XRE1, the closest XRE from the start codon in AHR2 gene, is mainly responsible for the binding with TCDD-activated AHR. This suggests that TCDD-activated AHR1 and AHR2 up-regulate the AHR2 mRNA levels and this auto-induced AHR2 may amplify the signal transduction of its downstream targets including CYP1A in the red seabream.

1,4-Dihydroxy-2-naphthoic acid (1,4-DHNA) is a bacterial-derived metabolite that binds the aryl hydrocarbon receptor (AhR) and exhibits anti-inflammatory activity in the gut. The structure-dependent AhR activity of hydroxyl/carboxy-substituted naphthoic acids (NAs) was determined in young adult mouse colonic (YAMC) cells and human Caco2 colon cancer cells using CYP1A1/CYP1B1 mRNAs as Ah-responsive genes. Compounds used in this study include 1,4-, 3,5-, and 3,7-DHNA, 1,4-dimethoxy-2-naphthoic acid (1,4-DMNA), 1- and 4-hydroxy-2-naphthoic acid (1-HNA, 4-HNA), 1- and 2-naphthoic acid (1-NA, 2-NA), and 1- and 2-naphthol (1-NOH, 2-NOH). 1,4-DHNA was the most potent compound among hydroxyl/carboxy naphthalene derivatives, and the fold induction response for CYP1A1 and CYP1B1 was similar to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in YAMC and Caco2 cells. 1- and 4-HNA were less potent than 1,4-DHNA but induced maximal (TCDD-like) response for CYP1B1 (both cell lines) and CYP1A1 (Caco2 cells). With the exception of 1- and 2-NA, all compounds significantly induced Cyp1b1 in YAMC cells and these responses were not observed in AhR-deficient YAMC cells generated using CRISPR/Cas9 technology. In addition, we also observed that 1- and 2-NOH (and 1,4-DHNA) were weak AhR agonists, and 1- and 2-NOH also exhibited partial AhR antagonist activity. Structure-activity relationship studies for CYP1A1 but not CYP1B1 were similar in both cell lines, and CYP1A1 induction required one or both 1,4-dihydroxy substituents and activity was significantly enhanced by the 2-carboxyl group. We also used computational analysis to show that 1,4-DHNA and TCDD share similar interactions within the AhR binding pocket and differ primarily due to the negatively charged group of 1,4-DHNA.

Laboratory safety requires protecting personnel from chemical exposures. Working with stock solutions of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDFs) in routine analysis of feed and food with bioanalytical or physicochemical methods raises some concerns. Since PCDD/PCDFs are considered as possibly acutely toxic, the potential risks were evaluated to determine whether supervision of their use is necessary. Based on LD50-data for oral or dermal intake, hazard classification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a substance (category 1) and in commercially available TCDD standard solutions (category 4) is different. As worst case exposure scenario during routine laboratory work it was assumed that a dose of 100 ng TCDD gets onto the skin and is absorbed. This would result in the total body burden of a 70 kg person with 15 kg fat increasing from 10 (upper range of current background levels) to similar to 17 pg of toxic equivalents (TEQs) of PCDD/PCDFs per g lipid, a level commonly observed over past decades. Chloracne, the main acute effect occurring weeks after exposure, is observed at much higher blood concentrations than estimated from accidental laboratory exposure. Immunotoxicity, developmental effects and other toxic effects may occur at lower blood levels, but require longer periods to develop. Since acute toxic symptoms don't occur within an "8 h acute time window", no supervision is necessary when working with standard solutions in routine analysis. Nevertheless, precautionary measures are needed regarding long-term adverse health effects and appropriate workplace conditions must exist to ensure that additional occupational exposure to PCDD/PCDFs by laboratory personnel is negligible.

The Ah receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and naturally-occurring chemicals. Although a significant amount of information is available with respect to the planar aromatic hydrocarbon AhR ligands, the actual spectrum of chemicals that can bind to and activate the AhR is only now being elucidated. In addition, the lack of information regarding the actual three-dimensional structure of the AhR ligand binding domain (LBD) has hindered detailed analysis of the molecular mechanisms by which these ligands bind to and active AhR signal transduction. In this review we describe the current state of knowledge with respect to naturally occurring AhR ligands and present and discuss the first theoretical model of the AhR LBD based on crystal structures of homologous PAS family members.

The Ah receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and naturally-occurring chemicals. Although a significant amount of information is available with respect to the planar aromatic hydrocarbon AhR ligands, the actual spectrum of chemicals that can bind to and activate the AhR is only now being elucidated. In addition, the lack of information regarding the actual three-dimensional structure of the AhR ligand binding domain (LBD) has hindered detailed analysis of the molecular mechanisms by which these ligands bind to and active AhR signal transduction. In this review we describe the current state of knowledge with respect to naturally occurring AhR ligands and present and discuss the first theoretical model of the AhR LBD based on crystal structures of homologous PAS family members.

BackgroundAssociation of particulate matter with adverse health effects has been established in epidemiological studies and animal experiments. Epidemiological studies are difficult to undertake while animal studies are impractical for high-throughput toxicity testing. The ease and rapidity of in vitro tests emphasizes their potential for use in risk assessment of chemicals and particles. We examined the association between in vitro and in vivo responses to ambient particles, to determine the potential of cell-based assays as standalone toxicity screening tools.MethodsAssays of cytotoxicity and key inflammatory mediators were applied to determine the in vitro biological potency of a panel of urban and mineral particles in J774A.1 macrophages and A549 lung epithelial cells. The particles were also screened for the presence of AhR agonists using the Ah receptor-dependent gene induction assay and for endotoxin using the Limulus amebocyte lysate assay. A subset of the particles with a contrasting in vitro toxicity profile was delivered intratracheally in BALB/c mice to assess their in vivo biological potency. Results from various bioassays were combined within the in vitro and in vivo models. The combined potency measures were examined for associations.ResultsOverall, J774A.1 cells were more sensitive to particle effects than A549 cells. Whereas the combined cytotoxicity estimates were highly correlated between the two cell lines, the combined in vitro inflammatory potency estimates were not, emphasizing functional differences of the two cell types. Secretion of inflammatory markers by J774A.1 cells was correlated with AhR ligand binding profile and endotoxin levels of particles. Particle instillation led to an acute toxicity response in BALB/c mice, with neutrophilia and release of inflammatory mediators. While the combined toxicity estimates were not correlated between in vitro and in vivo models, the combined inflammatory and integrated potency estimates (toxicity an

Fishes in estuarine waters are frequently exposed to treated wastewater effluent, among numerous other sources of contaminants, yet the impacts of these anthropogenic chemicals are not well understood in these dynamic and important waterways. Inland silversides (Menidia beryllina) at an early stage of development [12 days posthatch (dph)] were exposed to waters from two estuarine wastewater-treatment outfall locations in a tidal estuary, the Sacramento/San Joaquin Delta (California, USA) that had varied hydrology and input volumes. The genomic response caused by endocrine-disrupting compounds (EDCs) in these waters was determined using quantitative polymerase chain reaction on a suite of hormonally regulated genes. Relative androgenic and estrogenic activities of the waters were measured using CALUX reporter bioassays. The presence of bifenthrin, a pyrethroid pesticide and known EDC, as well as caffeine and the anti-inflammatory pharmaceutical ibuprofen, which were used as markers of wastewater effluent input, were determined using instrumental analysis. Detectable levels of bifenthrin (2.89 ng L(-1)) were found on one of the sampling dates, and caffeine was found on all sampling dates, in water from the Boynton Slough. Neither compound was detected at the Carquinez Strait site, which has a much smaller effluent discharge input volume relative to the receiving water body size compared with Boynton Slough. Water samples from both sites incubated in the CALUX cell line induced estrogenic and androgenic activity in almost all instances, though the estrogenicity was relatively higher than the androgenicity. Changes in the abundance of mRNA transcripts of endocrine-responsive genes and indicators of general chemical stress were observed after a 96-h exposure to waters from both locations. The relative levels of endocrine response, changes in gene transcript abundance, and contaminant concentrations were greater in water from the Boynton Slough site despite thos

Methyl-tert-butyl ether (MTBE) and its degradation by-product, tert-butyl alcohol (TBA), are widespread contaminants detected frequently in groundwater in California. Since MTBE was used as a fuel oxygenate for almost two decades, leaking underground fuel storage tanks are an important source of contamination. Gasoline components such as BTEX (benzene, toluene, ethylbenzene and xylenes) are often present in mixtures with MTBE and TBA. Investigations of interactions between BTEX and MTBE degradation have not yielded consistent trends, and the molecular mechanisms of BTEX compounds' impact on MTBE degradation are not well understood. We investigated trends in transcription of biodegradation genes in the MTBE-degrading bacterium, Methylibium petroleiphilum PM1 upon exposure to MTBE, TBA, ethylbenzene and benzene as individual compounds or in mixtures. We designed real-time quantitative PCR assays to target functional genes of strain PM1 and provide evidence for induction of genes mdpA (MTBE monooxygenase), mdpJ (TBA hydroxylase) and bmoA (benzene monooxygenase) in response to MTBE, TBA and benzene, respectively. Delayed induction of mdpA and mdpJ transcription occurred with mixtures of benzene and MTBE or TBA, respectively. bmoA transcription was similar in the presence of MTBE or TBA with benzene as in their absence. Our results also indicate that ethylbenzene, previously proposed as an inhibitor of MTBE degradation in some bacteria, inhibits transcription of mdpA, mdpJ and bmoAgenes in strain PM1.

BackgroundAssociation of particulate matter with adverse health effects has been established in epidemiological studies and animal experiments. Epidemiological studies are difficult to undertake while animal studies are impractical for high-throughput toxicity testing. The ease and rapidity of in vitro tests emphasizes their potential for use in risk assessment of chemicals and particles. We examined the association between in vitro and in vivo responses to ambient particles, to determine the potential of cell-based assays as standalone toxicity screening tools.MethodsAssays of cytotoxicity and key inflammatory mediators were applied to determine the in vitro biological potency of a panel of urban and mineral particles in J774A.1 macrophages and A549 lung epithelial cells. The particles were also screened for the presence of AhR agonists using the Ah receptor-dependent gene induction assay and for endotoxin using the Limulus amebocyte lysate assay. A subset of the particles with a contrasting in vitro toxicity profile was delivered intratracheally in BALB/c mice to assess their in vivo biological potency. Results from various bioassays were combined within the in vitro and in vivo models. The combined potency measures were examined for associations.ResultsOverall, J774A.1 cells were more sensitive to particle effects than A549 cells. Whereas the combined cytotoxicity estimates were highly correlated between the two cell lines, the combined in vitro inflammatory potency estimates were not, emphasizing functional differences of the two cell types. Secretion of inflammatory markers by J774A.1 cells was correlated with AhR ligand binding profile and endotoxin levels of particles. Particle instillation led to an acute toxicity response in BALB/c mice, with neutrophilia and release of inflammatory mediators. While the combined toxicity estimates were not correlated between in vitro and in vivo models, the combined inflammatory and integrated potency estimates (toxicity an

Malassezia furfur yeast strains isolated from diseased human skin preferentially biosynthesize indole alkaloids which can be detected in the human skin and are highly potent activators of the aryl hydrocarbon receptor (AhR) and AhR-dependent gene expression. Chemical analysis of an EtOAc extract of a M. furfur strain obtained from diseased human skin and grown on l-tryptophan agar revealed several known AhR active tryptophan metabolites along with a previously unidentified compound, pityriazepin. While its structure resembled that of the known alkaloid pityriacitrin, the comprised pyridine ring had been transformed into an azepinone. The indoloazepinone scaffold of pityriazepin is extremely rare in nature and has only been reported once previously. Pityriazepin, like the other isolated compounds, was found to be a potent activator of the AhR-dependent reporter gene assay in recombinant cell lines derived from four different species, although significant species differences in relative potency were observed. The ability of pityriazepin to competitively bind to the AhR and directly stimulate AhR DNA binding classified it as a new naturally-occurring potent AhR agonist. M. furfur produces an expanded collection of extremely potent naturally occurring AhR agonists, which produce their biological effects in a species-specific manner.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of structurally diverse chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). As part of a larger effort to identify the full spectrum of chemicals that can bind to and activate the AhR, we have examined the ability of several naturally occurring marine-derived brominated indoles and brominated (methylthio)indoles (collectively referred to as brominated indoles) to bind to the AhR and stimulate AhR-dependent gene expression. Incubation of mouse, rat, and guinea pig recombinant cell lines containing a stably transfected AhR-responsive luciferase reporter gene with eight brominated indoles revealed that all compounds stimulated luciferase reporter gene activity, although some species-specific differences were observed. All compounds induced significantly more luciferase activity when incubated with cells for 4 h as compared to 24 h, demonstrating that these compounds are transient activators of the AhR signaling pathway. Three of the brominated indoles induced CYP1A1 mRNA in human HepG2 cells in vitro and Cyp1a mRNA in zebrafish embryos in vivo. The identification of the brominated indoles as direct ligands and activators/agonists of the AhR was confirmed by their ability to compete with [(3)H]TCDD for binding to the AhR and to stimulate AhR transformation and DNA binding in vitro. Taken together, these results indicate that marine-derived brominated indoles are members of a new class of naturally occurring AhR agonists.

Among the few bacteria known to utilize methyl tert-butyl ether (MTBE) as a sole carbon source, Methylibium petroleiphilum PM1 is a well-characterized organism with a sequenced genome; however, knowledge of the genetic regulation of its MTBE degradation pathway is limited. We investigated the role of a putative transcriptional activator gene, mdpC, in the induction of MTBE-degradation genes mdpA (encoding MTBE monooxygenase) and mdpJ (encoding tert-butyl alcohol hydroxylase) of strain PM1 in a gene-knockout mutant mdpC(-). We also utilized quantitative reverse transcriptase PCR assays targeting genes mdpA, mdpJ and mdpC to determine the effects of the mutation on transcription of these genes. Our results indicate that gene mdpC is involved in the induction of both mdpA and mdpJ in response to MTBE and tert-butyl alcohol (TBA) exposure in PM1. An additional independent mechanism may be involved in the induction of mdpJ in the presence of TBA.

Small molecular weight protein kinase inhibitors are frequently used tools to unravel the complex network of cellular signal transduction under certain physiological and pathophysiological conditions. 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) is a widely used compound to block the activity of Src family kinases, the major group of non-receptor tyrosine kinases, which trigger multiple cellular signaling pathways. Here, we show that PP2 induces cytochrome P450 1A1 mRNA expression and enzyme activity in a dose-dependent manner in human HepG2 hepatoma cells and NCTC 2544 keratinocytes. By means of reporter gene assays, RNA interference, electrophoretic mobility shift assay, and competitive ligand-binding assay, we further demonstrate that PP2 is a ligand for the aryl hydrocarbon receptor (AHR), an intracellular chemosensor that regulates xenobiotic metabolism, environmental stress responses, and immune functions. Upon ligand-dependent activation, the AHR translocates into the nucleus and dimerizes with the AHR nuclear translocator (ARNT) to modulate the expression of its target genes. In addition, AHR activation is frequently accompanied by an activation of the tyrosine kinase c-Src, resulting in stimulation of cell-surface receptors and downstream signal transduction. As PP2 activates the AHR/ARNT pathway by simultaneously blocking c-Src-mediated alternative signaling routes, this compound may be a suitable tool to study the contribution of the different AHR-dependent signaling pathways to biological processes and adverse outcomes. On the other hand, the unexpected property of PP2 to stimulate AHR/ARNT signaling should be carefully taken into account in future investigations in order to avoid a false interpretation of experimental results and molecular interrelations.

The Ah receptor (AhR)-responsive CALUX (chemically activated luciferase expression) cell bioassay is commonly used for rapid screening of samples for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), dioxin-like compounds, and AhR agonists/antagonists. By increasing the number of AhR DNA recognition sites (dioxin responsive elements), we previously generated a novel third generation (G3) recombinant AhR-responsive mouse CALUX cell line (H1L7.5c3) with a significantly enhanced response to DLCs compared to existing AhR-CALUX cell bioassays. However, the elevated background luciferase activity of these cells and the absence of comparable G3 cell lines derived from other species have limited their utility for screening purposes. Here, we describe the development and characterization of species-specific G3 recombinant AhR-responsive CALUX cell lines (rat, human, and guinea pig) that exhibit significantly improved limit of detection and dramatically increased TCDD induction response. The low background luciferase activity, low minimal detection limit (0.1 pM TCDD) and enhanced induction response of the rat G3 cell line (H4L7.5c2) over the H1L7.5c3 mouse G3 cells, identifies them as a more optimal cell line for screening purposes. The utility of the new G3 CALUX cell lines were demonstrated by screening sediment extracts and a small chemical compound library for the presence of AhR agonists. The improved limit of detection and increased response of these new G3 CALUX cell lines will facilitate species-specific analysis of DLCs and AhR agonists in samples with low levels of contamination and/or in small sample volumes.

Among the few bacteria known to utilize methyl tert-butyl ether (MTBE) as a sole carbon source, Methylibium petroleiphilum PM1 is a well-characterized organism with a sequenced genome; however, knowledge of the genetic regulation of its MTBE degradation pathway is limited. We investigated the role of a putative transcriptional activator gene, mdpC, in the induction of MTBE-degradation genes mdpA (encoding MTBE monooxygenase) and mdpJ (encoding tert-butyl alcohol hydroxylase) of strain PM1 in a gene-knockout mutant mdpC(-). We also utilized quantitative reverse transcriptase PCR assays targeting genes mdpA, mdpJ and mdpC to determine the effects of the mutation on transcription of these genes. Our results indicate that gene mdpC is involved in the induction of both mdpA and mdpJ in response to MTBE and tert-butyl alcohol (TBA) exposure in PM1. An additional independent mechanism may be involved in the induction of mdpJ in the presence of TBA.

The Ah receptor (AhR)-responsive CALUX (chemically activated luciferase expression) cell bioassay is commonly used for rapid screening of samples for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), dioxin-like compounds, and AhR agonists/antagonists. By increasing the number of AhR DNA recognition sites (dioxin responsive elements), we previously generated a novel third generation (G3) recombinant AhR-responsive mouse CALUX cell line (H1L7.5c3) with a significantly enhanced response to DLCs compared to existing AhR-CALUX cell bioassays. However, the elevated background luciferase activity of these cells and the absence of comparable G3 cell lines derived from other species have limited their utility for screening purposes. Here, we describe the development and characterization of species-specific G3 recombinant AhR-responsive CALUX cell lines (rat, human, and guinea pig) that exhibit significantly improved limit of detection and dramatically increased TCDD induction response. The low background luciferase activity, low minimal detection limit (0.1 pM TCDD) and enhanced induction response of the rat G3 cell line (H4L7.5c2) over the H1L7.5c3 mouse G3 cells, identifies them as a more optimal cell line for screening purposes. The utility of the new G3 CALUX cell lines were demonstrated by screening sediment extracts and a small chemical compound library for the presence of AhR agonists. The improved limit of detection and increased response of these new G3 CALUX cell lines will facilitate species-specific analysis of DLCs and AhR agonists in samples with low levels of contamination and/or in small sample volumes.

Malassezia furfur yeast strains isolated from diseased human skin preferentially biosynthesize indole alkaloids which can be detected in the human skin and are highly potent activators of the aryl hydrocarbon receptor (AhR) and AhR-dependent gene expression. Chemical analysis of an EtOAc extract of a M. furfur strain obtained from diseased human skin and grown on l-tryptophan agar revealed several known AhR active tryptophan metabolites along with a previously unidentified compound, pityriazepin. While its structure resembled that of the known alkaloid pityriacitrin, the comprised pyridine ring had been transformed into an azepinone. The indoloazepinone scaffold of pityriazepin is extremely rare in nature and has only been reported once previously. Pityriazepin, like the other isolated compounds, was found to be a potent activator of the AhR-dependent reporter gene assay in recombinant cell lines derived from four different species, although significant species differences in relative potency were observed. The ability of pityriazepin to competitively bind to the AhR and directly stimulate AhR DNA binding classified it as a new naturally-occurring potent AhR agonist. M. furfur produces an expanded collection of extremely potent naturally occurring AhR agonists, which produce their biological effects in a species-specific manner.

Small molecular weight protein kinase inhibitors are frequently used tools to unravel the complex network of cellular signal transduction under certain physiological and pathophysiological conditions. 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) is a widely used compound to block the activity of Src family kinases, the major group of non-receptor tyrosine kinases, which trigger multiple cellular signaling pathways. Here, we show that PP2 induces cytochrome P450 1A1 mRNA expression and enzyme activity in a dose-dependent manner in human HepG2 hepatoma cells and NCTC 2544 keratinocytes. By means of reporter gene assays, RNA interference, electrophoretic mobility shift assay, and competitive ligand-binding assay, we further demonstrate that PP2 is a ligand for the aryl hydrocarbon receptor (AHR), an intracellular chemosensor that regulates xenobiotic metabolism, environmental stress responses, and immune functions. Upon ligand-dependent activation, the AHR translocates into the nucleus and dimerizes with the AHR nuclear translocator (ARNT) to modulate the expression of its target genes. In addition, AHR activation is frequently accompanied by an activation of the tyrosine kinase c-Src, resulting in stimulation of cell-surface receptors and downstream signal transduction. As PP2 activates the AHR/ARNT pathway by simultaneously blocking c-Src-mediated alternative signaling routes, this compound may be a suitable tool to study the contribution of the different AHR-dependent signaling pathways to biological processes and adverse outcomes. On the other hand, the unexpected property of PP2 to stimulate AHR/ARNT signaling should be carefully taken into account in future investigations in order to avoid a false interpretation of experimental results and molecular interrelations.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of structurally diverse chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). As part of a larger effort to identify the full spectrum of chemicals that can bind to and activate the AhR, we have examined the ability of several naturally occurring marine-derived brominated indoles and brominated (methylthio)indoles (collectively referred to as brominated indoles) to bind to the AhR and stimulate AhR-dependent gene expression. Incubation of mouse, rat, and guinea pig recombinant cell lines containing a stably transfected AhR-responsive luciferase reporter gene with eight brominated indoles revealed that all compounds stimulated luciferase reporter gene activity, although some species-specific differences were observed. All compounds induced significantly more luciferase activity when incubated with cells for 4 h as compared to 24 h, demonstrating that these compounds are transient activators of the AhR signaling pathway. Three of the brominated indoles induced CYP1A1 mRNA in human HepG2 cells in vitro and Cyp1a mRNA in zebrafish embryos in vivo. The identification of the brominated indoles as direct ligands and activators/agonists of the AhR was confirmed by their ability to compete with [(3)H]TCDD for binding to the AhR and to stimulate AhR transformation and DNA binding in vitro. Taken together, these results indicate that marine-derived brominated indoles are members of a new class of naturally occurring AhR agonists.

For a better understanding of species-specific relative effect potencies (REPs), responses of dioxin-like compounds (DLCs) were assessed. REPs were calculated using chemical-activated luciferase gene expression assays (CALUX) derived from guinea pig, rat, and mouse cell lines. Almost all 20 congeners tested in the rodent cell lines were partial agonists and less efficacious than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For this reason, REPs were calculated for each congener using concentrations at which 20% of the maximal TCDD response was reached,(REP20TCDD). REP20TCDD values obtained for PCDD/Fs were comparable with their toxic equivalency factors assigned by the World Health Organization (WHO-TEF), while those for PCBs were in general lower than the WHO-TEF values. Moreover, the guinea pig cell line was the most sensitive as indicated by the 20% effect concentrations of TCDD of 1.5, 5.6, and 11.0 pM for guinea pig, rat, and mouse cells, respectively. A similar response pattern was observed using multivariate statistical analysis between the three CALLTX assays and the WHO-TEFs. The mouse assay showed minor deviation due to higher relative induction potential for 2,3,7,8-tetrachlorodibenzofuran and 2,3,4,6,7,8-hexachlorodibenzofuran and lower for 1,2,3,4,6,7,8-heptachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl (PCB126). 2,3,7,8-Tetrachlorodibenzofuran was more than two times more potent in the mouse assay as compared with that of rat and guinea pig cells, while measured REP20TCDD for PCB126 was lower in mouse cells (0.05) as compared with that of the guinea pig (0.2) and rat (0.07). In order to provide REP20TCDD values for all WHO-TEF assigned compounds, quantitative structure activity relationship (QSAR) models were developed. The QSAR models showed that specific electronic properties and molecular surface characteristics play important roles in the AhR-mediated response. In silica derived REP20TCDD values were generally consistent with the WHO-TEFs w

The ceramic toximeter as a passive sampler in combination with the CALUX bioassay was utilized as a time-integrated pollution-assessment technique for dioxin-like PCDD/Fs in wastewaters. Toximeters filled with XCARB and enclosed in stainless steel cages were submerged in wastewater of Belbeis drainage-canal and EMAK paper-mill, located in Egypt, for 28 days. Two samplers were removed every week from each site. Extracts from toximeters, from bottom-sediments, and from paper-mill sludge were analyzed by the CALUX. Results showed a regular increase in the amounts of PCDD/Fs trapped by the toximeters as sampling time increased. Time-weighted average concentrations (TWA) of PCDD/Fs in the wastewaters of the drainage-canal and paper-mill were 231 (214-281) and 26 (24.1-32.6) pg-BEQL(-1), respectively. Compared to literature data, this means a high pollution level for the drainage-canal water, while the pollution level in the paper-mill wastewater is similar to that reported for other paper mills. The PCDD/Fs levels in the drainage-canal bottom-sediments and in the paper-mill sludge were 8.3 and 14.6 pg-BEQg(-1) dry-weight, at the lower end of internationally reported values in similar compartments. The sampling rate of the tested toximeters for dissolved PCDD/Fs was about 3.6 mL d(-1). Sediment/water partitioning coefficient logKd of PCDD/Fs in the drainage-canal and the paper-mill was 1.42 and 2.70 respectively. The organic-carbon normalized partition coefficient logKoc was 4.17 and 3.19 respectively, and is lower than the reported values for other sites.

Ceramic passive samplers or toximeters (packed with active carbon 1%, w/w, on celite), in combination with the CALUX bioassay have been used as a time-integrated monitoring technique for dissolved dioxin-like PCBs in urban and industrial wastewaters. The technique showed to be reliable during laboratory experiments: (1) PCB-126 amounts extracted from the passive samplers increased linearly with the time of exposure and (2) PCB-126 concentrations calculated from the amounts accumulated by the passive samplers were in agreement with their concentrations in the testing solution. Afterwards the toximeters were applied in the field. Two sampling sites located in Egypt were chosen: the Belbeis drainage canal, and the EMAK paper mill. A total of 18 ceramic toximeters were exposed to the wastewater in both sampling sites for a maximum period of 4 weeks. Two samplers were collected weekly from each site to monitor the increase in target analytes over time. Extracts were analyzed using the CALUX bioassay and the total dioxin-like PCB toxicity was reported for the aqueous phase (water column), as well as the solid phase (sediment and sludge) in both sampling sites. The time-weighted average concentration (TWA) of dl-PCBs in wastewater of the paper mill during the sampling period ranged between 7.1 and 9.1 pg-BEQL(-1), while that of the drainage canal ranged between 9.5 and 12.2 pg-BEQL(-1). The dl-PCBs in the fibrous sludge (paper mill) and bottom sediment (drainage canal) were 0.5 and 0.4 pg-BEQg(-1) dry-weight, respectively. The organic-carbon normalized partition coefficients between sediment and water (logKoc) for the paper mill and the canal were 2.4 and 4.3, respectively.

Azole antifungal ketoconazole (KET) was demonstrated to activate aryl hydrocarbon receptor (AhR). Since clinically used KET is a racemic mixture of two cis-enantiomers (2R,4S)-(+)-KET and (2S,4R)-(-)-KET, we examined the effects of KET enantiomers on AhR signaling pathway. (+)-KET dose-dependently activated AhR in human gene reporter cell line AZ-AHR, and displayed 5-20× higher agonist activity (efficacy), as compared to (-)-KET; both enantiomers were AhR antagonists with equal potency (IC50). Consistently, (+)-KET strongly induced CYP1A1 mRNA and protein in human HepG2 cells, while (-)-KET exerted less than 10% of (+)-KET activity. In primary human hepatocytes, both enantiomers preferentially induced CYP1A2 over CYP1A1 mRNA and protein, and the potency of (+)-KET was slightly higher as compared to (-)-KET. Ligand binding assay with guinea pig liver cytosols revealed that both (+)-KET and (-)-KET are weak ligands of AhR that displaced [3H]-TCDD with comparable potency. Similarly, both enantiomers weakly transformed AhR to DNA-binding form with similar potency, as showed by EMSA, in guinea pig liver cytosolic extracts and nuclear extracts from mouse Hepa-1 cells. We also examined effects of KET on glucocorticoid receptor (GR), a regulator of AhR activity. Both KET enantiomers antagonized GR with similar potency, as revealed by gene reporter assay in AZ-GR cell line and down-regulation of tyrosine aminotransferase mRNA in human hepatocytes. Finally, we demonstrate enantiospecific antifungal activities of KET enantiomers in six Candida spp. strains. In conclusion, the significance of current study is providing the first evidence of enatiospecific effects of cis-enantiomers of ketoconazole on AhR-CYP1A pathway.

Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.