Cytosolic Aryl Hydrocarbon Receptor Research Articles

Abstract 6882: Exploring aryl hydrocarbon receptor expression and distribution in the tumor microenvironment, with a focus on immune cells, in various solid cancer types

Abstract Background: The aryl hydrocarbon receptor (AhR) is a transcription factor that performs various biological functions upon ligand activation. While there have been extensive studies and development of AhR-targeting anticancer therapies, including active clinical trials, research on the expression of AhR, a key biomarker, in the TME remain limited. Herein, we evaluated the nuclear and cytosolic AhR expression across five solid cancer types: head and neck squamous cell carcinoma (HNSCC), bladder cancer, colorectal cancer, esophageal cancer, and non-small cell lung cancer (NSCLC). Methods: Multiplexed immunohistochemistry (mIHC) and image cytometry were employed to investigate the AhR expression and distribution in 513 patient samples, of which 292 were from patients with one of five solid cancer types. For quantification of AhR expression levels, FCS files were generated, and the resulting data was analyzed using the R program. Results: AhR expression was predominantly high in cancer cells, followed by T cells and macrophages across all cancer types. All 513 patient samples were categorized into three clusters based on distinct expression and localization patterns of AhR. Cluster 1 exhibited high AhR expression in the cancer cell nucleus, whereas cluster 3 showed the lowest. AhR expression in the stromal macrophage nucleus was the most pronounced in cluster 2. In cluster 1, NSCLC was entirely absent, with esophageal cancer representing the largest proportion. In contrast, cluster 2 was notably different from cluster 1, with a 5% prevalence of NSCLC and a relatively higher proportion of HNSCC and colorectal cancer. The AhR expression in macrophages within cluster 2 was noticeably higher than in cluster 1. Cluster 3, which had the highest NSCLC proportion, generally displayed low AhR expression compared to other clusters, but also exhibited AhR expression in regulatory T cells (Tregs), a pattern which was not observed in other cancer types. Notably, a positive correlation was observed between the nuclear and cytosolic expressions of AhR, suggesting that AhR expression as a biomarker is independent of its subcellular localization. Furthermore, there was no clear correlation between AhR expression levels and cancer stage, grade, and metastatic status. Conclusion: We elucidated the expression profile of AhR within the TME across five cancer types, then classified the patient samples into three clusters based on their distinct AhR expression patterns. By demonstrating different expression patterns of AhR in cancer cells and immune cells, our findings are anticipated to provide a fundamental basis for clinical and immunological research on AhR-targeting therapies. Citation Format: Dong Kwon Kim, Chai Young Lee, Yu Jin Han, So Young Park, Heekyung Han, Kwangmin Na, Mi Hyun Kim, Seung Min Yang, Sujeong Baek, Youngtaek Kim, Joon Yeon Hwang, Seul Lee, Seong-san Kang, Taedong Han, Hyounmie Doh, Jongho Cho, Dajeong Kim, Daewon Cha, Min Hee Hong, Sun Min Lim, Jii Bum Lee, Jae Hwan Kim, Kyoung-Ho Pyo, Byoung Chul Cho. Exploring aryl hydrocarbon receptor expression and distribution in the tumor microenvironment, with a focus on immune cells, in various solid cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6882.

Tyrosol: Repercussion of the Lack of a Hydroxyl-Group in the Response of MCF-7 Cells to Hypoxia.

In solid tumors, such as breast cancer, hypoxic microenvironment worsens patient prognoses. We have previously reported in MCF-7 breast cancer cells that, under hypoxic conditions, hydroxytyrosol (HT) downregulates the level of reactive oxygen species, reduces the expression of hypoxia inducible factor-1α (HIF-1α), and, at high concentrations, can bind to the aryl hydrocarbon receptor (AhR). With this background, the present study investigated whether the most abundant extra virgin olive oil (EVOO) phenolic compound tyrosol (TYR), with a chemical structure similar to HT but with only one hydroxyl group, exerts comparable effects. Our results revealed that, although TYR did not show any antioxidant activity in hypoxic MCF-7 cells, it inhibited the PI3K/Akt/mTOR/S6 kinase (S6K) pathway and reduced the expression of HIF-1α and some of its target genes. Besides, TYR showed a lower binding affinity with the cytosolic transcription factor AhR, and even reduced its transcriptional activity. Some of these results are positive to control tumor progression in a hypoxic environment; however, they are observed at doses unachievable with diet intake or nutraceutical presentations. Considering that EVOO phenols can have synergistic effects, a mixture of low doses of TYR and other phenols could be useful to achieve these beneficial effects.

Cryo-EM structure of the cytosolic AhR complex

Aryl hydrocarbon receptor (AhR) is an important ligand-activated transcription factor involved in the regulation of various important physiological functions. Here, we report the cryo-EM structures of the Hsp90-AhR-p23 complex with or without bound XAP2, where the structure of the mouse AhR PAS-B domain is resolved. A highly conserved bridge motif of AhR is responsible for the interaction with the Hsp90 dimeric lumen. The ligand-free AhR PAS-B domain is attached to the Hsp90 dimer and is stabilized in the complex with bound XAP2. In addition, the DE-loop and a group of conserved pocket inner residues in the AhR PAS-B domain are found to be important for ligand binding. These results reveal the structural basis of the biological functions of AhR. Moreover, the protein purification method presented here allows the isolation of stable mouse AhR protein, which could be used to develop high-sensitivity biosensors for environmental pollutant detection.

Chlamydia trachomatis relies on the scavenger role of aryl hydrocarbon receptor with detyrosinated tubulin for its intracellular growth, but this is impaired by excess indole

Although IFN-γ depletes tryptophan (Trp) as a defense against intracellular Chlamydia trachomatis (Ct) infected to hypoxic vagina, the presence of indole, a precursor of Trp, enables Ct to infect IFN-γ-exposed culture cells. Meanwhile, Trp-derived indole derivatives interact the aryl hydrocarbon receptor (AhR), which is a ligand-dependent transcription factor involved in the cellular homeostasis with tubulin dynamics. Here, the amounts of IFN-γ and indole in cervical swabs with known Ct infection status were measured, and Ct growth in the presence of indole was determined from the perspective of the AhR axis under hypoxia. A positive correlation between the amounts of IFN-γ and indole was found, and both of these amounts were lower in Ct-positive swabs than in Ct-negative ones. Indole as well as other AhR ligands inhibited Ct growth, especially under normoxia. Ct prompted the expression of detyrosinated tubulin (dTTub), but indole inhibited it. Indole did not stimulate the translocation of AhR to nucleus, and it blocked AhR activation in AhR-reporter cells. Ct growth was reduced more effectively under normoxia in AhR-knockdown cells, an effect that was enhanced by indole, which in turn diminished dTTub. Thus, Ct growth relies on the scavenger role of cytosolic AhR responsible for promoting dTTub expression.

Tyrphostin AG1024 downregulates aryl hydrocarbon receptor (AhR) expression in an IGF1R and IR-independent manner

The aryl hydrocarbon receptor (AhR) is a receptor-type transcription factor that is crucial for endocrine disruption and carcinogenesis caused by environment chemicals. Previous studies have indicated that certain intracellular signals are involved in AhR activation by their agonists, but the detailed mechanism remains unclear. In this study, we screened for important molecules for AhR activation using SCAD inhibitor kits. Among 164 kinase inhibitors listed in these kits, tyrphostin AG1024, commonly used as an inhibitor of insulin-like growth factor receptor (IGF1R) and insulin receptor (IR), was identified as a potent inhibitor of 3-methylcholanthrene (MC)-mediated AhR activation. We further investigated the mechanism by which AG1024 suppresses MC-mediated AhR activation. AG1024 decreased AhR-dependent luciferase activity, CYP1A1 gene expression, and its protein expression. However, when IGF1R siRNA and IR siRNA were used, AhR activation was slightly increased, in contrast to AG1024 treatment. In addition, AG1024 treatment downregulated the expression of AhR protein but not AhR gene, and decreased both nucleic and cytosolic AhR proteins. Therefore, AG1024 suppressed AhR activation by downregulating AhR protein expression. The molecular target of AG1024 remains unclear, and should be an important target for the regulation of AhR-dependent toxicity.

Theoretical investigation of AhR binding property with relevant structural requirements for AhR-mediated toxicity of polybrominated diphenyl ethers

Polybrominated diphenyl ethers (PBDEs) are more frequently suspected with the induction of toxicity via signal transduction pathway of cytosolic aryl hydrocarbon receptor (AhR), the initial binding to which is assumed to be an essential prerequisite during the ligand-dependent activation. However, the AhR binding property and associated toxicity of PBDEs is yet to be clearly known for lacking insights into the structural requirements at molecular level. To understand the AhR binding property of PBDEs, the ligand binding domain (LBD) of AhR was simulatively developed on homologous protein after basic validation of geometrical rationality and the binding interaction profile was visually described using molecular docking approach. For AhR binding, the offset or edge-on π-π stackings with aromatic motifs including Phe289, Phe345 and His285 were shown to be structurally required whereas the electrostatic attraction validated for AhR binding to dioxins might be less effective for 2,2′,3,4,4′-pentabromodiphenyl ether (BDE-85). Besides the demands of less steric hindrance from alanines and weak formulation of hydrogen bonds, the dispersion force through large contact and polarization of S-π electrons seemed to be impactful when BDE-85 were closer to Cys327, Met334 or Met342. With theoretical computation of AhR binding energies, the more significant correlativity with bioassays was derived especially for the lowly/moderately brominated congeners, and could be used to predict the AhR binding affinity on certain degree. The informative results would thus not only help well understand the molecular basis of AhR-mediated toxicity but give an approach for accelerative evaluation of AhR binding and toxicity of PBDEs.

Abstract PR15: The oncometabolite R-2-Hydroxyglutarate suppresses the innate immune microenvironment of IDH1-mutated gliomas via aryl hydrocarbon receptor signaling

Abstract Background: IDH1-mutated gliomas are associated with less abundant and phenotypically skewed innate and adaptive immune cell infiltrates compared to IDH1 wild-type tumors. Despite this, the most frequent mutation—IDH1R132H—represents a clonal shared neoantigen and mutations in IDH are associated with a more favorable prognosis. While the tumor cell-intrinsic consequences of the oncometabolite R-2-hydroxyglutarate (R-2-HG) accumulating in IDH1-mutated gliomas as a result of a neomorphic enzymatic function are well-characterized, potential direct paracrine effects of R-2-HG influencing the glioma immune microenvironment remain incompletely understood. Aim: This study aimed at characterizing the impact of the oncometabolite R-2-HG on the innate immune microenvironment of IDH1-mutated gliomas. Methods and Results: By means of comprehensive analyses of expression datasets from human gliomas and syngeneic murine tumor models as well as transporter studies, we demonstrate that R-2-HG is imported by both microglia and macrophages via SLC family transporters and suppresses their function in a paracrine manner. Functional analyses of microglia and macrophages indicate an R-2-HG-driven induction of tolerogenicity as evidenced by accumulation of IL10 and TGFβ and suppression of MHC-II expression, which results in impaired activation of antigen-specific T-cells and activation of immune checkpoint molecules. Multilevel signature profiling of human tumor-infiltrating as well as primary immune cells was complemented by reporter gene assays and pathway analyses and revealed that R-2-HG activates the cytosolic transcription factor aryl hydrocarbon receptor (AHR), a key immunomodulatory target of immunosuppressive tryptophan metabolism. By means of knockout models, the observed immunosuppressive phenotype was shown to be AHR-dependent. Functional relevance of R-2-HG-mediated, AHR-driven impairment of myeloid cell immunity was demonstrated in vivo by pharmacologic AHR inhibition, increasing the efficacy of checkpoint blockade. Conclusion: R-2-HG impairs antitumor immunity in IDH1-mutated gliomas by activating the AHR in innate immune cells, thus suppressing the innate immune microenvironment by compromising antigen presentation and activation of antigen-specific T-cells. This, together with recent findings on inhibitory effects on T-cell immunity, represents a novel mechanism of immune evasion of an immunogenic driver mutation and opens a novel therapeutic approach to IDH1-mutated gliomas. Citation Format: Mirco Friedrich, Lukas Bunse, Theresa Bunse, Edward Green, Tobias Kessler, Stefan Pusch, Katrin Deumelandt, Rafael Carretero, Andreas von Deimling, Francisco J Quintana, Wolfgang Wick, Michael Platten. The oncometabolite R-2-Hydroxyglutarate suppresses the innate immune microenvironment of IDH1-mutated gliomas via aryl hydrocarbon receptor signaling [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR15.

Inhibition of pancreatic cancer Panc1 cell migration by omeprazole is dependent on aryl hydrocarbon receptor activation of JNK

Several aryl hydrocarbon receptor (AhR)-active pharmaceuticals were screened as inhibitors of pancreatic cancer cell invasion and identified two compounds, omeprazole, that inhibited invasion. Inhibition of highly invasive Panc1 cell invasion by omeprazole involves an AhR-dependent non-genomic pathway, and omeprazole-mediated inhibition of Panc1 cell invasion was dependent on Jun-N-terminal kinase (JNK) and mitogen-activated kinase kinase 7 (MKK7). The failure of omeprazole to induce nuclear translocation of the AhR was not due to overexpression of cytosolic AhR partner proteins Hsp90 or XAP2, and results of DNA sequencing show that the AhR expressed in Panc1 cells was not mutated. Results of RNAseq studies indicate that omeprazole induced an AhR-dependent downregulation of several pro-invasion factors including activated leukocyte cell adhesion molecule (ALCAM), long chain fatty acid CoA-synthase (CSL4), stathmin 3 (STMN3) and neuropillin 2 (NRP2), and the specific functions of these genes are currently being investigated.

Mitochondrial-targeted aryl hydrocarbon receptor and the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cellular respiration and the mitochondrial proteome

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor within the Per-Arnt-Sim (PAS) domain superfamily. Exposure to the most potent AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is associated with various pathological effects including metabolic syndrome. While research over the last several years has demonstrated a role for oxidative stress and metabolic dysfunction in AHR-dependent TCDD-induced toxicity, the role of the mitochondria in this process has not been fully explored. Our previous research suggested that a portion of the cellular pool of AHR could be found in the mitochondria (mitoAHR). Using a protease protection assay with digitonin extraction, we have now shown that this mitoAHR is localized to the inter-membrane space (IMS) of the organelle. TCDD exposure induced a degradation of mitoAHR similar to that of cytosolic AHR. Furthermore, siRNA-mediated knockdown revealed that translocase of outer-mitochondrial membrane 20 (TOMM20) was involved in the import of AHR into the mitochondria. In addition, TCDD altered cellular respiration in an AHR-dependent manner to maintain respiratory efficiency as measured by oxygen consumption rate (OCR). Stable isotope labeling by amino acids in cell culture (SILAC) identified a battery of proteins within the mitochondrial proteome influenced by TCDD in an AHR-dependent manner. Among these, 17 proteins with fold changes≥2 are associated with various metabolic pathways, suggesting a role of mitochondrial retrograde signaling in TCDD-mediated pathologies. Collectively, these studies suggest that mitoAHR is localized to the IMS and AHR-dependent TCDD-induced toxicity, including metabolic dysfunction, wasting syndrome, and hepatic steatosis, involves mitochondrial dysfunction.

Functional analysis of the dioxin response elements (DREs) of the murine CYP1A1 gene promoter: beyond the core DRE sequence.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological and toxicological effects of halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). When activated by dioxin, the cytosolic AhR protein complex translocates into the nucleus and dimerizes with the ARNT (Ah receptor nuclear translocator) protein. The heteromeric ligand:AhR/Arnt complex then recognizes and binds to its specific DNA recognition site, the dioxin response element (DRE). DREs are located upstream of cytochrome P4501A1 (CYP1A1) and other AhR-responsive genes, and binding of the AhR complex stimulates their transcription. Although CYP1A1 expression has been used as the model system to define the biochemical and molecular mechanism of AhR action, there is still limited knowledge about the roles of each of the seven DREs located in the CYP1A1 promoter. These seven DREs are conserved in mouse, human and rat. Deletion analysis showed that a single DRE at −488 was enough to activate the transcription. Truncation analysis demonstrated that the DRE at site −981 has the highest transcriptional efficiency in response to TCDD. This result was verified by mutation analysis, suggesting that the conserved DRE at site −981 could represent a significant and universal AhR regulatory element for CYP1A1. The reversed substituted intolerant core sequence (5′-GCGTG-3′ or 5′-CACGC-3′) of seven DREs reduced the transcriptional efficiency, which illustrated that the adjacent sequences of DRE played a vital role in activating transcription. The core DRE sequence (5′-TNGCGTG-3′) tends to show a higher transcriptional level than that of the core DRE sequence (5′-CACGCNA-3′) triggered by TCDD. Furthermore, in the core DRE (5′-TNGCGTG-3′) sequence, when “N” is thymine or cytosine (T or C), the transcription efficiency was stronger compared with that of the other nucleotides. The effects of DRE orientation, DRE adjacent sequences and the nucleotide “N” in the core DRE (5′-TNGCGTG-3′) sequence on the AhR-regulated CYP1A1 transcription in response to TCDD were studied systematically, and our study laid a good foundation for further investigation into the AhR-dependent transcriptional regulation triggered by dioxin and dioxin-like compounds.

Nucleotide specificity of DNA binding of the aryl hydrocarbon receptor:ARNT complex is unaffected by ligand structure.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and a wide variety of structurally diverse ligands through its ability to translocate into the nucleus and bind to a specific DNA recognition site (the dioxin-responsive element [DRE]) adjacent to responsive genes. Although the sequence of the DRE is well defined, several reports suggested that the nucleotide specificity of AhR DNA binding may vary depending on the structure of its bound ligand. Given the potential toxicological significance of this hypothesis, an unbiased DNA-selection-and-PCR-amplification approach was utilized to directly determine whether binding and activation of the AhR by structurally diverse agonists alter its nucleotide specificity of DNA binding. Guinea pig hepatic cytosolic AhR activated in vitro by equipotent concentrations of TCDD, 3-methylcholanthrene, β-naphthoflavone, indirubin, L-kynurenine, or YH439 was incubated with a pool of DNA oligonucleotides containing a 15-base pair variable region consisting of all possible nucleotides. The AhR-bound oligonucleotides isolated by immunoprecipitation were PCR amplified and used in subsequent rounds of selection. Sequence analysis of a total of 196 isolated oligonucleotides revealed that each ligand-activated AhR:ARNT complex only bound to DRE-containing DNA oligonucleotides; no non-DRE-containing DNA oligonucleotides were identified. These results demonstrate that the binding and activation of the AhR by structurally diverse agonists do not appear to alter its nucleotide specificity of DNA binding and suggest that stimulation of gene expression mediated by direct DNA binding of ligand-activated AhR:ARNT complexes is DRE dependent.

Ginsenosides are novel naturally-occurring aryl hydrocarbon receptor ligands.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of structurally diverse chemicals. In this study, we examined the ability of a series of ginsenosides extracted from ginseng, a traditional Chinese medicine, to bind to and activate/inhibit the AHR and AHR signal transduction. Utilizing a combination of ligand and DNA binding assays, molecular docking and reporter gene analysis, we demonstrated the ability of selected ginsenosides to directly bind to and activate the guinea pig cytosolic AHR, and to stimulate/inhibit AHR-dependent luciferase gene expression in a recombinant guinea pig cell line. Comparative studies revealed significant species differences in the ability of ginsenosides to stimulate AHR-dependent gene expression in guinea pig, rat, mouse and human cell lines. Not only did selected ginsenosides preferentially activate the AHR from one species and not others, mouse cell line was also significantly less responsive to these chemicals than rat and guinea pig cell lines, but the endogenous gene CYP1A1 could still be inducted in mouse cell line. Overall, the ability of these compounds to stimulate AHR signal transduction demonstrated that these ginsenosides are a new class of naturally occurring AHR agonists.

Sunitinib, a tyrosine kinase inhibitor, induces cytochrome P450 1A1 gene in human breast cancer MCF7 cells through ligand-independent aryl hydrocarbon receptor activation

Sunitinib (SUN) is a new multi-targeted oral tyrosine kinase inhibitor that has both anti-angiogenic and anti-tumor activities. However, information reported in the literature on the effects of SUN on the constitutive expression of cytochrome P450 1A1 (CYP1A1) gene in cells from mammalian species remains unclear. Therefore, the main objectives of the current work were to investigate the potentiality of SUN to induce CYP1A1 gene expression in human breast cancer MCF7 cells and to explore the molecular mechanisms involved. Our results showed that SUN induced the CYP1A1 mRNA, protein, and activity levels in a concentration-dependent manner in MCF7 cells. The increase in CYP1A1 mRNA by SUN was completely blocked by the transcriptional inhibitor, actinomycin D; implying that SUN increased de novo RNA synthesis. Furthermore, the ability of SUN to increase luciferase reporter gene expression suggests an aryl hydrocarbon receptor (AhR)-dependent transcriptional control and excludes the possibility of any posttranscriptional mechanisms. In addition, blocking of AhR activation by resveratrol, a well-known AhR antagonist, prevented the SUN-induced CYP1A1 gene expression, further confirms the involvement of AhR. Interestingly, this was associated with the inability of SUN to directly bind to and induce transformation of cytosolic AhR to its DNA-binding form in vitro, suggesting that the effect of SUN does not involve direct binding to AhR. The current manuscript provides the first evidence for the ability of SUN to induce CYP1A1 gene expression in MCF7 cells through AhR ligand-independent mechanisms.

Fused mesoionic heterocyclic compounds are a new class of aryl hydrocarbon receptor (AhR) agonist of exceptional potency

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent ligands of the aryl hydrocarbon receptor (AhR). Here, we show that a novel fused mesoionic heterocyclic compound (AZ1) is ∼5-fold more potent than TCDD in both rat and human cell lines at inducing cytochrome P4501A1 RNA. In rat H4IIE cells, AZ1 gave an EC50=5.05pM (95% CI=2.81–9.09pM) whereas TCDD had an EC50=25.5pM (95% CI=18.2–36.0pM). AZ1 was also more potent than TCDD (5–10-fold) at inducing the AhR-related CYP1A2 and CYP1B1 genes, showing that AZ1 is more potent at inducing multiple genes. In human MCF-7 cells AZ1 gave an EC50=65.4pM (95% CI=45.6–93.7pM) and TCDD an EC50=241pM (95% CI=161–362pM), showing that AZ1 was more potent than TCDD at inducing CYP1A1 RNA in multiple species. Finally, the compound bound to rat cytosolic AhR with 6-fold higher affinity than TCDD, showing that the highly potent agonism of this substance is mediated via a high affinity for the receptor. This data shows that this novel compound, which shares structural similarities with various naphthoflavones, is a potent ligand of the AhR.

An Evaluation of Single Nucleotide Polymorphisms in the Human Heat Shock Protein 90 kDa Alpha and Beta Isoforms

Single nucleotide polymorphisms (SNPs) in genes coding for proteins that maintain the cytosolic aryl hydrocarbon receptor (AHR) complex may affect individual susceptibility to dioxin-like compound (DLC)-induced toxicity. The cytosolic 90 kDa heat shock proteins (HSP90s) are ubiquitous chaperone proteins that bind to and stabilize numerous client proteins, including non-ligand-bound AHR. The objective of this study was to characterize SNPs in the human cytosolic HSP90 genes (HSP90AA1 and HSP90AB1). DNA sequencing of 101 human samples detected eight and seven unique SNPs at the HSP90AA1 and HSP90AB1 loci, respectively. For HSP90AA1, two non-synonymous (L71M and E554D) and one rare early termination (Q107X) SNP were observed. One SNP (E554D) was a rare novel polymorphism located in the middle substrate binding region. All SNPs detected in the HSP90AB1 gene were synonymous. With the exception of Q107X, in silico analyses predicted all HSP90 SNPs would have very low to medium risk of affecting the regulation of alternative splicing in gene transcription or protein function. Overall, a very limited presence of SNPs with predicted functional consequence in key domains of the human HSP90 proteins was observed in this study.

Inhibition of Heme Oxygenase-1 Partially Reverses the Arsenite-Mediated Decrease of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 Catalytic Activity in Isolated Rat Hepatocytes

Heme oxygenase (HO-1), the rate-limiting enzyme in the physiological breakdown of heme, is ubiquitous, and its expression can be increased by arsenite [As(III)], and similar other stimuli that induce cellular oxidative stress. Interestingly, it has been shown that the As(III)-induced HO-1 is inversely correlated with a decrease in cytochromes P450 (P450s) activity; however, the direct role for HO-1 in the inhibition of P450 enzymes remains unknown. Our results showed that As(III) at a concentration of 5 μM decreased the constitutive and inducible expression of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 at the mRNA, protein, and catalytic activity levels. Moreover, As(III) decreased the nuclear accumulation of aryl hydrocarbon receptor (AhR) and pregnane X receptor without increasing their degradation. As(III) also increased the binding of cytosolic AhR to heat shock protein 90 and hepatitis B virus X-associated protein 2. In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin as an inducer for CYP1A and rifampin as an inducer for CYP3A, As(III) decreased the enzymatic activity of the four P450s more than it decreased their mRNA or protein expression levels. It is noteworthy that treatment with the competitive HO-1 inhibitor, tin-mesoporphyrin, or supplementing external heme partially reversed the As(III)-mediated decrease in activities of the four P450s. In conclusion, the current study provides the first evidence that As(III) decreases CYP1A1, CYP1A2, CYP3A23, and CYP3A2 expression in freshly isolated rat primary hepatocytes. Furthermore, inhibiting the As(III)-mediated induction of HO-1 partially restores the enzymatic activity of these P450s that was initially decreased by As(III), confirming the direct role of HO-1 in the inhibition of P450s.

Predictive insight into the relationship between AhR binding property and toxicity of polybrominated diphenyl ethers by PLS-derived QSAR

Polybrominated diphenyl ethers (PBDEs) are experimentally indicated to be capable of binding to the cytosolic aryl hydrocarbon receptor (AhR) and show a weak or moderate toxicity. However, little is yet known about the AhR-mediated toxicology. To fully evaluate the structural effects of PBDE ligand on AhR binding affinity and toxicity, quantitative structure–activity relationships (QSARs) were developed by PLS analysis. In this study, a simple but potent QSAR that was qualified with much better or comparable performance of prediction was optimally established for PBDE toxicity. With QSAR analysis, the AhR binding property was carefully described to reflect the origin of AhR binding affinity. Besides the effects from topological characters, the dispersion and electrostatic interactions were of indispensability for AhR binding affinity whereas the dispersion was further suggested to be dominant. The structural requirement for AhR binding affinity and toxicity was also investigated. As was similarly observed for polychlorinated biphenyls (PCBs), the preferential bromination at para- and meta (particularly 3,3′-)-sites was confirmed as a key determinant to improve the AhR binding affinity and the toxicity of PBDEs.

Another Important Biological Function for the Aryl Hydrocarbon Receptor

For many years, the aryl hydrocarbon receptor (AhR) had been studied primarily for its contribution to environmental chemical-induced organ toxicity or carcinogenicity.1–3 The prevailing paradigm fashioned from these studies held that activation of the cytosolic AhR by any one of a variety of environmental pollutants (eg, planar polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and dioxins) results in nuclear AhR translocation and transcriptional upregulation of prototypic target genes encoding cytochrome P450 enzymes (eg, CYP1A1 and CYP1B1).4–6 These enzymes are capable of metabolizing at least some of the environmental AhR ligands into toxic or mutagenic intermediates, thereby affecting biological outcomes. Although these studies were extremely important for defining the AhR as an environmental ligand-induced transcription factor and in mapping out portions of the AhR signaling pathway, they did not directly provide evidence of the “normal” physiological function of …

Dioxins and Human Toxicity

The term dioxins usually refers to polychlorinated dibenzo-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). As 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) has the highest toxic potential, the toxic potentials of other PCDDs and PCDFs are defined in comparison with it. Human exposure to dioxins can be environmental (background), occupational, or accidental pollution. In the human body, dioxins are in part metabolised and eliminated, and the rest is stored in body fat. People vary in their capacity to eliminate TCDD, but it is also dose-dependent; the elimination rate is much faster at higher than lower levels. The liver microsomal P4501A1 enzyme oxygenates lipophilic chemicals such as dioxins. It is encoded by the CYP1A1 gene. Cytosolic aryl hydrocarbon receptor (AhR) mediates their carcinogenic action. It binds to dioxin, translocates to nucleus and together with hydrocarbon nuclear translocator (ARNT) and xenobiotic responsive element (XRE) increases the expression of CYP1A1.Dioxins are classified as known human carcinogens, but they also cause noncancerous effects like atherosclerosis, hypertension, and diabetes. Long-term exposures to dioxins cause disruption of the nervous, immune, reproductive, and endocrine system. Short-term exposure to high levels impairs the liver function and causes chloracne. The most sensitive population to dioxin exposure are the foetuses and infants.A large number of health effects have been documented in the scientific literature, and they all place dioxins among the most toxic chemicals known to man.

The Aryl Hydrocarbon Receptor-interacting Protein (AIP) Is Required for Dioxin-induced Hepatotoxicity but Not for the Induction of the Cyp1a1 and Cyp1a2 Genes

The aryl hydrocarbon receptor (AHR) plays an essential role in the toxic response to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), in the adaptive up-regulation of xenobiotic metabolizing enzymes, and in hepatic vascular development. In our model of AHR signaling, the receptor is found in a cytosolic complex with a number of molecular chaperones, including Hsp90, p23, and the aryl hydrocarbon receptor-interacting protein (AIP), also known as ARA9 and XAP2. To understand the role of AIP in adaptive and toxic aspects of AHR signaling, we generated a conditional mouse model where the Aip locus can be deleted in hepatocytes. Using this model, we demonstrate two important roles for the AIP protein in AHR biology. (i) The expression of AIP in hepatocytes is essential to maintain high levels of functional cytosolic AHR protein in the mammalian liver. (ii) Expression of the AIP protein is essential for dioxin-induced hepatotoxicity. Interestingly, classical AHR-driven genes show differential dependence on AIP expression. The Cyp1b1 and Ahrr genes require AIP expression for normal up-regulation by dioxin, whereas Cyp1a1 and Cyp1a2 do not. This differential dependence on AIP provides evidence that the mammalian genome contains more than one class of AHR-responsive genes and suggests that a search for AIP-dependent, AHR-responsive genes may guide us to the targets of the dioxin-induced hepatotoxicity.