An aryl hydrocarbon receptor agonist suppresses the growth of human umbilical vein endothelial cells in vitro: Potent effect with polyunsaturated fatty acids.

Human umbilical vein endothelial cells (HUVECs) are a pivotal component of the hematopoietic microenvironment linked to the modulation of the immune response, inflammation and carcinogenesis. HUVEC expresses the aryl hydrocarbon receptor (AHR), which regulates gene expression by binding to the xenobiotic-responsive element. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent agonist for AHR signalling. Treatment with TCDD (0.1-100 nmol/L) was found to suppress the proliferation and to stimulate the death of HUVEC. TCDD's effects were abolished by culturing with CH223191, an inhibitor of AHR signalling. Mechanistically, TCDD treatment increased the protein levels of cell growth suppressors, including p53, Rb, p21 and regucalcin, and caspase-3 implicated in apoptotic cell death, and decreased the levels of Stat3, mitogen-activated protein kinase (MAPK/Erk1/2) and phospho-MAPK/Erk1/2. Treatment with polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid, eicosapentaenoic acid and arachidonic acid, suppressed the proliferation and stimulated the death of HUVEC in vitro, and decreased the levels of Stat3, MAPK/Erk1/2 and phospho-MAPK/Erk1/2 and increased caspase-3. Notably, the effects of TCDD in suppressing proliferation and stimulating death of HUVEC were modulated by coculturing with PUFAs. These effects were reversed by treatment with CH223191, an inhibitor of AHR. Treatment with both TCDD and PUFAs collaboratively enhanced the levels of AHR, CYP1A1, p53, p21, Rb and regucalcin. Moreover, TCDD suppressed migration with wound healing of HUVEC. Notably, the combination of TCDD and PUFAs revealed potent suppressive effects on angiogenesis of HUVEC, potentially related to disorders of the stromal microenvironment.

Aryl Hydrocarbon Receptor-Dependent inductions of omega-3 and omega-6 polyunsaturated fatty acid metabolism act inversely on tumor progression.

The Western diet contains a high ratio of omega-6 (ω6) to omega-3 (ω3) polyunsaturated fatty acids (PUFA). The prototypical aryl hydrocarbon receptor (AHR) ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), induces CYP1 family enzymes, which can metabolize PUFA to epoxides. Mice fed ω3-rich or ω6-rich diets were treated with TCDD and injected subcutaneously with AHR-competent Hepa1-GFP hepatoma cells or AHR-deficient LLC lung cancer cells. TCDD reduced the growth rates of the resulting tumors in ω3-fed mice and inhibited their metastasis to the liver and/or lung, but had the opposite effects in mice fed ω6 PUFA. These responses were likely attributable to the corresponding PUFA epoxides generated in tumor cells and/or host, since many depended upon co-administration of a soluble epoxide hydrolase (EPHX2) inhibitor in males, and/or were associated with increases in epoxide levels in tumors and sites of metastasis. Equivalent effects occurred in females in the absence of EPHX2 inhibition, probably because this sex expressed reduced levels of EPHX2. The responses elicited by TCDD were associated with effects on tumor vascularity, tumor cell proliferation and/or apoptosis. Thus environmental AHR agonists, and potentially also endogenous, nutritional, and microbiome-derived agonists, may reduce or enhance cancer progression depending on the composition of dietary PUFA, particularly in females.

A CRISPR/Cas9 Whole-Genome Screen Identifies Genes Required for Aryl Hydrocarbon Receptor-Dependent Induction of Functional CYP1A1.

Environmental pollutants including halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, including benzo[a]pyrene, exert their deleterious effects through the activation of the aryl hydrocarbon receptor (AHR) and by the resulting transcription of genes not yet fully identified. Ligand-bound AHR translocates from cytoplasm to nucleus, where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. The AHR/ARNT dimer binds to enhancer regions of responsive genes to activate transcription. AHR also mediates carcinogenesis caused by PAHs, likely via CYP1A1, CYP1A2, and CYP1B1, which are massively induced by activated AHR in many tissues and generate carcinogenic electrophilic derivatives of PAHs. In the current study, we have used the mouse GeCKOv2 genome-wide CRISPR/Cas9 library to identify novel genes in the AHR pathway by taking advantage of a B[a]P selection assay that we previously used to identify core AHR pathway genes in Hepa-1c1c7 murine hepatoma cells. Besides Ahr, Arnt, and Cyp1a1, we report the identification of multiple additional putative AHR pathway genes including several that we validated. These include cytochrome P450 reductase (Por), which mediates redox regeneration of cytochromes P450, and 5 genes of the heme biosynthesis pathway: delta-aminolevulinate synthase 1 (Alas1), porphobilinogen deaminase (Hmbs), uroporphyrinogen decarboxylase (Urod), coproporphyrinogen oxidase (Cpox), and ferrochelatase (Fech): heme being an essential prosthetic group of cytochrome P450 proteins. Notably, several of these genes were identified by GeCKO screening, despite not being identifiable by reverse genetics approaches. This indicates the power of high-sensitivity genome-wide genetic screening for identifying genes in the AHR pathway.

2,3,7,8­tetrachlorodibenzo­p­dioxin suppresses the growth of human colorectal cancer cells in vitro: Implication of the aryl hydrocarbon receptor signaling.

Human colorectal cancer is the third most common cancer disease with a 5­year survival rate of 55% in USA in 2016. The investigation to identify novel biomarker factors with molecular classification may provide notable clinical information to prolong the survival of patients with colorectal cancer. The aryl hydrocarbon receptor (AHR) binds the AHR nuclear translocator in the cytoplasm of various types of cells, including liver cells, and then binds to the xenobiotic responsive element on various genes. AHR was initially discovered via its ligand, the polychlorinated hydrocarbon, 2,3,7,8­tetrachlorodibenzo­p­dioxin (TCDD). The present study was undertaken to determine whether TCDD, an agonist of AHR signaling, impacts the growth of RKO human colorectal cancer cells in vitro. Treatment with TCDD (0.1­100 nM) revealed suppressive effects on colony formation and proliferation of RKO cells, and stimulated death of these cells with subconfluence. These effects of TCDD were abolished by pretreatment with CH223191, an inhibitor of AHR signaling. Western blot analysis demonstrated that TCDD treatment decreased AHR levels and elevated cytochrome P450 family 1 subfamily A member 1 (CYP1A1) levels, indicating a stimulation of AHR signaling. TCDD treatment caused an increase in nuclear factor­κB p65 and ß­catenin levels, although it did not have an effect on Ras levels. Notably, TCDD treatment increased the levels of p53, retinoblastoma, p21 and regucalcin, which are depressors of carcinogenesis. Additionally, action of TCDD on cell proliferation and death were not revealed in regucalcin­overexpressing RKO cells, and regucalcin overexpression depressed AHR signaling associated with CYP1A1 expression. Thus, AHR signaling suppresses the growth of colorectal cancer cells, indicating a role as a significant targeting molecule for colorectal cancer.

Prolonged survival of renal cancer patients is concomitant with a higher regucalcin gene expression in tumor tissues: Overexpression of regucalcin suppresses the growth of human renal cell carcinoma cells in vitro.

Renal cell carcinoma (RCC), which is a type of cancer found in the kidney tubule, is among the 10 most frequently occurring human cancers. Regucalcin plays a potential role as a regulator of transcriptional activity, and its downregulated expression or activity may contribute to the promotion of human cancers. In this study, we investigated the involvement of regucalcin in human RCC. Regucalcin expression was compared in 23 normal and 29 tumor samples of kidney cortex tissues of patients with clear cell RCC obtained through the Gene Expression Omnibus (GEO) database (GSE36895). Regucalcin expression was downregulated in the tumor tissues. The prolonged survival of patients with clear cell RCC was demonstrated to be associated with a higher regucalcin gene expression in the TCGA dataset. The overexpression of regucalcin suppressed the colony formation, proliferation and the death of human clear cell RCC A498 cells in vitro. Mechanistically, the overexpression of regucalcin induced the G1 and G2/M phase cell cycle arrest of A498 cells through the suppression of multiple signaling components, including Ras, PI3 kinase, Akt and mitogen­activated protein (MAP) kinase. Importantly, the overexpression of regucalcin led to an elevation in the levels of the tumor suppressors, p53, Rb and the cell cycle inhibitor, p21. The levels of the transcription factors, c­fos, c­jun, nuclear factor­κB p65, ß­catenin and signal transducer and activator of transcription 3, were suppressed by regucalcin overexpression. On the whole, the findings of this study suggest that regucalcin plays a suppressive role in the promotion of human RCC. The overexpression of regucalcin by gene delivery systems may thus prove to be a novel therapeutic strategy for RCC.

2,3,7,8­Tetrachlorodibenzo­p­dioxin suppresses the growth of human liver cancer HepG2 cells in vitro: Involvement of cell signaling factors.

The aryl hydrocarbon receptor (AHR) is transcriptionally active in the form of a heterodimer with the AHR nuclear translocator, which then binds to the xenobiotic responsive element. AHR was originally discovered via its ligand, the polychlorinated hydrocarbon, 2,3,7,8­tetrachlorodibenzo­p­dioxin (TCDD). In this study, we investigated whether TCDD regulates the growth of human liver cancer HepG2 cells in vitro. TCDD (0.1­100 nM) was found to exert suppressive effects on the colony formation and proliferation of HepG2 cells, and stimulatory effects on the death of HepG2 cells when the cells reached subconfluence. The effects of TCDD on the HepG2 cells were abolished by culture with CH223191, an inhibitor of AHR signaling. The effects of TCDD were dependent on the concentration of serum, which contains various signaling factors. The effects of TCDD were not potentiated by culture with tumor necrosis factor­α, which activates the signaling of nuclear factor­κB (NF­κB). The results of western blot analysis revealed that TCDD increased the protein levels of p53, Rb, p21, and regucalcin, which are suppressors of the growth of tumor cells. Moreover, TCDD enhanced the NF­κB p65, ß­catenin, signal transducer and activator of transcription 3 (STAT3), Ras and Akt levels. Thus, the findings of this study indicate that TCDD may suppress liver cancer cell growth through various signaling pathways, mediated by AHR and its­related co­factors. Of note, the effects of TCDD were found to be potentiated by gemcitabine, which induces nuclear DNA damage in cancer cells, suggesting that their combined use may have potential as a suppressor of tumor cell growth.

ChIP-re-ChIP: Co-occupancy Analysis by Sequential Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) exploits the specific interactions between DNA and DNA-associated proteins. It can be used to examine a wide range of experimental parameters. A number of proteins bound at the same genomic location can identify a multi-protein chromatin complex where several proteins work together to regulate gene transcription or chromatin configuration. In many instances, this can be achieved using sequential ChIP; or simply, ChIP-re-ChIP. Whether it is for the examination of specific transcriptional or epigenetic regulators, or for the identification of cistromes, the ability to perform a sequential ChIP adds a higher level of power and definition to these analyses. In this chapter, we describe a simple and reliable method for the sequential ChIP assay.

Role of diets rich in omega-3 and omega-6 in the development of cancer / Papel de las dietas ricas en omega-3 y omega-6 en el desarrollo del cáncer

Abstract: Over the past decade, some studies have addressed the therapeutic effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) and the opposite effects of omega-6 (ω-6) PUFAs on several diseases, including cardiovascular disorders, diabetes, neurodegenerative diseases, and cancer. Research demonstrates the safety of these naturally occurring ingredients. Of particular interest, several studies have shown that ω-3 PUFAs possess a therapeutic role against certain types of cancer. It is also known that ω-3 PUFAs can improve the efficacy and tolerability of chemotherapy. Previous reports have indicated that suppression of nuclear factor-κB, activation of AMPK/SIRT1, modulation of cyclooxygenase (COX) activity, and up-regulation of novel anti-inflammatory lipid mediators such as protectins, maresins, and resolvins, are the main mechanisms of the antineoplastic effect of ω-3 PUFAs. In contrast, several studies have demonstrated that ω-6 PUFAs induce progression in certain types of cancer. In this review, we discuss epidemiological and experimental studies addressing the relationship between the development of some types of cancer, including colon and colorectal carcinoma, breast cancer, prostate cancer, lung cancer and neuroblastoma, and the ingestion to ω-3 and ω-6 (PUFAs). We also discuss the clinical data, addressing the therapeutic role of omega-3 PUFA against different types of cancer.

Resumen: Durante las últimas décadas algunos estudios se han enfocado en los efectos terapéuticos de los ácidos grasos poliinsaturados (AGPI) omega-3 (ω-3) y los efectos contrarios de los AGPI omega-6 (ω-6) en diversas enfermedades, incluyendo enfermedades cardiovasculares, diabetes, enfermedades neurodegenerativas y cáncer. Las investigaciones han mostrado la seguridad de estos lípidos naturales. En particular, varios estudios han mostrado que los AGPI ω-3 poseen un efecto terapéutico contra ciertos tipos de cáncer. También se sabe que los AGPI ω-3 pueden mejorar la eficacia y tolerancia de la quimioterapia. En publicaciones anteriores se ha indicado que la supresión del factor nuclear κB, la activación de AMPK/SIRT1, la modulación de la actividad de la ciclooxigenasa (COX) y la regulación positiva de nuevos mediadores lipídicos antiinflamatorios como las protectinas, maresinas y resolvinas, son los principales mecanismos del efecto antineoplásico de los AGPI ω-3. En contraste, otros estudios han demostrado que los AGPI ω-6 inducen la progresión de ciertos tipos de cáncer. En esta revisión se discuten algunos estudios experimentales y epidemiológicos que abordan la relación entre el desarrollo de algunos tipos de cáncer, como carcinoma de colon y colorrectal, cáncer de mama, próstata y pulmón, así como neuroblastoma, y la ingestión de AGPI ω-3 y ω-6. Así mismo, se discuten los datos clínicos sobre el papel terapéutico del AGPI ω-3 contra diferentes tipos de cáncer.

The role of AHR-inducible cytochrome P450s in metabolism of polyunsaturated fatty acids.

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) is the prototype of a large number of non-genotoxic carcinogens, dietary phytochemicals and endogenous metabolites that act via binding the aryl hydrocarbon receptor (AHR). The TCDD-liganded AHR massively upregulates CYP1A1, CYP1A2 and CYP1B1 in many mammalian organs. We demonstrated that TCDD treatment markedly increases the levels of several epoxides and diol metabolites of the epoxides of both ω-6 and ω-3 polyunsaturated fatty acids (PUFA) in the liver and lungs of mice, in an aryl hydrocarbon receptor-dependent fashion, and most likely via the activities of the CYP1 family members. ω-6 Epoxides are known to stimulate tumor growth, angiogenesis, and metastasis in mice. Interestingly, ω-3 epoxides have the opposite effect on these parameters. TCDD and other AHR agonists may, therefore, impact angiogenesis, growth and metastasis of tumors in either a positive or negative way, depending on the relative levels of ω -6 epoxides and ω-3 epoxides generated in the host and/or tumor cells. This is of potential relevance to carcinogenesis by AHR agonists in the human, since the human population is exposed to widely varying ω-6: ω-3 PUFA ratios in the diet.

Role of diets rich in omega-3 and omega-6 in the development of cancer.

Over the past decade, some studies have addressed the therapeutic effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) and the opposite effects of omega-6 (ω-6) PUFAs on several diseases, including cardiovascular disorders, diabetes, neurodegenerative diseases, and cancer. Research demonstrates the safety of these naturally occurring ingredients. Of particular interest, several studies have shown that ω-3 PUFAs possess a therapeutic role against certain types of cancer. It is also known that ω-3 PUFAs can improve the efficacy and tolerability of chemotherapy. Previous reports have indicated that suppression of nuclear factor-κB, activation of AMPK/SIRT1, modulation of cyclooxygenase (COX) activity, and up-regulation of novel anti-inflammatory lipid mediators such as protectins, maresins, and resolvins, are the main mechanisms of the antineoplastic effect of ω-3 PUFAs. In contrast, several studies have demonstrated that ω-6 PUFAs induce progression in certain types of cancer. In this review, we discuss epidemiological and experimental studies addressing the relationship between the development of some types of cancer, including colon and colorectal carcinoma, breast cancer, prostate cancer, lung cancer and neuroblastoma, and the ingestion to ω-3 and ω-6 (PUFAs). We also discuss the clinical data, addressing the therapeutic role of omega-3 PUFA against different types of cancer.

SIN3A, generally regarded as a transcriptional repressor, is required for induction of gene transcription by the aryl hydrocarbon receptor.

CYP1A1 bioactivates several procarcinogens and detoxifies several xenobiotic compounds. Transcription of CYP1A1 is highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the aryl hydrocarbon receptor. We recently described an RNAi high throughput screening performed in the Hepa-1 mouse hepatoma cell line, which revealed that SIN3A is necessary for the induction of CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) enzymatic activity by TCDD. In the current studies, we sought to provide insight into the role of SIN3A in this process, particularly because studies on SIN3A have usually focused on its repressive activity on transcription. We report that ectopic expression of human SIN3A in Hepa-1 cells enhanced EROD induction by TCDD and efficiently rescued TCDD induction of EROD activity in cells treated with an siRNA to mouse SIN3A, thus validating a role for SIN3A in CYP1A1 induction. We demonstrate that SIN3A is required for TCDD induction of the CYP1A1 protein in Hepa-1 cells but not for expression of the aryl hydrocarbon receptor protein. In addition, siRNAs for SIN3A decreased TCDD-mediated induction of CYP1A1 mRNA and EROD activity in human hepatoma cell line Hep3B. We establish that TCDD treatment of Hepa-1 cells rapidly increases the degree of SIN3A binding to both the proximal promoter and enhancer of the Cyp1a1 gene and demonstrate that increased binding to the promoter also occurs in human Hep3B, HepG2, and MCF-7 cells. These studies establish that SIN3A physically interacts with the CYP1A1 gene and extends the transcriptional role of SIN3A to a gene that is very rapidly and dramatically induced.

Treatment of mice with 2,3,7,8-Tetrachlorodibenzo-p-dioxin markedly increases the levels of a number of cytochrome P450 metabolites of omega-3 polyunsaturated fatty acids in the liver and lung.

We previously reported that 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increased the levels of several cytochrome P450 metabolites of the omega-6 polyunsaturated fatty acids (PUFAs), arachidonic acid (ARA) and linoleic acid in the serum, liver, lung and spleen of C57BL/6 mice in an aryl hydrocarbon receptor (AHR)-dependent fashion. These increases correlated with increased levels of CYP1A1, CYP1A2 and/or CYP1B1. In the current study, we measured 77 oxylipins, including 59 that we had not measured previously, and demonstrate that TCDD also markedly increases the levels of many epoxide and diol metabolites of the omega-3 PUFAs, α-linolenic acid, eicosapentaenoic acid (EPA) and docasahexaenoic acid (DHA) in these mice. Since these epoxide metabolites have been reported to have opposite effects on angiogenesis, tumor growth and tumor metastasis compared with the equivalent metabolites of omega-6 PUFA, these observations have important implications with regard to the potential involvement of the cytochrome P450 metabolites of PUFAs in mediating the biological effects of TCDD and other agonists of AHR.

Genome-wide RNAi high-throughput screen identifies proteins necessary for the AHR-dependent induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The aryl hydrocarbon receptor (AHR) has a plethora of physiological roles, and upon dysregulation, carcinogenesis can occur. One target gene of AHR encodes the xenobiotic and drug-metabolizing enzyme CYP1A1, which is inducible by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the AHR. An siRNA library targeted against over 5600 gene candidates in the druggable genome was used to transfect mouse Hepa-1 cells, which were then treated with TCDD, and subsequently assayed for CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) activity. Following redundant siRNA activity (RSA) statistical analysis, we identified 93 hits that reduced EROD activity with a p value ≤ .005 and substantiated 39 of these as positive hits in a secondary screening using endoribonuclease-prepared siRNAs (esiRNAs). Twelve of the corresponding gene products were subsequently confirmed to be necessary for the induction of CYP1A1 messenger RNA by TCDD. None of the candidates were deficient in aryl hydrocarbon nuclear translocator expression. However 6 gene products including UBE2i, RAB40C, CRYGD, DCTN4, RBM5, and RAD50 are required for the expression of AHR as well as for induction of CYP1A1. We also found 2 gene products, ARMC8 and TCF20, to be required for the induction of CYP1A1, but our data are ambiguous as to whether they are required for the expression of AHR. In contrast, SIN3A, PDC, TMEM5, and CD9 are not required for AHR expression but are required for the induction of CYP1A1, implicating a direct role in Cyp1a1 transcription. Our methods, although applied to Cyp1a1, could be modified for identifying proteins that regulate other inducible genes.

Potential role of epigenetic mechanisms in the regulation of drug metabolism and transport.

This is a report of a symposium on the potential role of epigenetic mechanisms in the control of drug disposition sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2013 meeting in Boston, MA, April 21, 2013. Epigenetics is a rapidly evolving area, and recent studies have revealed that expression of drug-metabolizing enzymes and transporters is regulated by epigenetic factors, including histone modification, DNA methylation, and noncoding RNAs. The symposium speakers provided an overview of genetic and epigenetic mechanisms underlying variable drug metabolism and drug response, as well as the implications for personalized medicine. Considerable insight into the epigenetic mechanisms in differential regulation of the dioxin-inducible drug and carcinogen-metabolizing enzymes CYP1A1 and 1B1 was provided. The role of noncoding microRNAs in the control of drug metabolism and disposition through targeting of cytochrome P450 (P450) enzymes and ATP-binding cassette membrane transporters was discussed. In addition, potential effects of xenobiotics on chromatin interactions and epigenomics, as well as the possible role of long noncoding RNAs in regulation of P450s during liver maturation were presented.

HIF-1 expression is associated with CCL2 chemokine expression in airway inflammatory cells: implications in allergic airway inflammation.

BACKGROUND: The pathogenesis of allergic airway inflammation in asthmatic patients is complex and characterized by cellular infiltrates and activity of many cytokines and chemokines. Both the transcription factor hypoxia inducible factor-1 (HIF-1) and chemokine CCL2 have been shown to play pivotal roles in allergic airway inflammation. The interrelationship between these two factors is not known. We hypothesized that the expression of HIF-1 and CCL2 may be correlated and that the expression of CCL2 may be under the regulation of HIF-1. Several lines of evidence are presented to support this hypothesis. METHODS: The effects of treating wild-type OVA (ovalbumin)-sensitized/challenged mice with ethyl-3,4-dihydroxybenzoate (EDHB), which upregulate HIF, on CCL2 expression, were determined. Mice conditionally knocked out for HIF-1ß was examined for their ability to mount an allergic inflammatory response and CCL2 expression in the lung after intratracheal exposure to ovalbumin. The association of HIF-1α and CCL2 levels was also measured in endobronchial biopsies and bronchial fluid of asthma patients after challenge. RESULTS: We show that both HIF-1α and CCL2 were upregulated during an OVA (ovalbumin)-induced allergic response in mice. The levels of HIF-1α and CCL2 were significantly increased following treatment with a pharmacological agent which upregulates HIF-1α, ethyl-3,4-dihydroxybenzoate (EDHB). In contrast, the expression levels of HIF-1α and CCL2 were decreased in the lungs of mice that have been conditionally knocked out for ARNT (HIF-1ß) following sensitization with OVA when compared to levels in wild type mice. In asthma patients, the levels of HIF-1α and CCL2 increased after challenge with the allergen. CONCLUSIONS: These data suggest that CCL2 expression is regulated, in part, by HIF-1 in the lung. These findings also demonstrate that both CCL2 and HIF-1 are implicated in the pathogenesis of allergic airway inflammation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment alters eicosanoid levels in several organs of the mouse in an aryl hydrocarbon receptor-dependent fashion.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many mammalian organs and tissues. These effects are mediated by the aryl hydrocarbon receptor (AHR). CYP1A1, CYP1A2 and CYP1B1 are upregulated by the liganded AHR. These (and other) cytochromes P450 can metabolize arachidonic acid into a variety of bioactive eicosanoids. Towards investigating a potential role of eicosanoids in TCDD toxicity, arachidonic acid, two other unsaturated long-chain fatty acids, and up to twenty-five eicosanoids were measured in five organs/tissues of male and female wild-type and Ahr null mice treated or untreated with TCDD. TCDD generally increased the levels of the four dihydroxyeicosatrienoic acids (DHETs) and (where measured) 5,6-epoxyeicosatrienoic acid and 18-, 19- and 20-hydroxyeicosatrienoic acids (HETEs) in the serum, liver, spleen and lungs, but not the heart, of both sexes, and increased the levels in the serum, liver and spleen of several metabolites that are usually considered products of lipoxygenase activity, but which may also be generated by cytochromes P450. TCDD also increased the levels of the esterified forms of these eicosanoids in the liver in parallel with the corresponding free forms. The levels of prostanoids were generally not affected by TCDD. The above changes did not occur in Ahr null mice, and are therefore mediated by the AHR. TCDD increased the mRNA levels of Cyp1a1, Cyp1a2, Cyp1b1 and the Pla2g12a form of phospholipase A(2) to varying degrees in the different organs, and these increases correlated with some but not all the changes in eicosanoids levels in the organs, suggesting that other enzymes may also be involved.

CYP2S1 is negatively regulated by corticosteroids in human cell lines.

Cytochrome P450s are monooxygenase proteins involved in the metabolism of both exogenous and endogenous compounds. CYP2S1 can metabolize eicosanoids in the absence of both NADPH and NADPH cytochrome P450 reductase, and can also activate the anticancer agent 1 AQ4N [1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxy anthracene-9,10-dione]. CYP2S1 is mainly expressed in extrahepatic tissues such as the trachea, lung, stomach, small intestine, spleen, skin, breast, kidney and placenta. Furthermore, increased expression of CYP2S1 occurs in several tumors of epithelial origin, making the characterization of CYP2S1 regulation relevant to the treatment of disease. We report that the synthetic glucocorticoid receptor ligand dexamethasone (DEX) represses CYP2S1 expression. The ED(50) is between 1 nM and 3 nM and maximal repression is reached by 48 h. Other corticosteroids are also effective at repressing CYP2S1. We show that repression by DEX is mediated by the glucocorticoid receptor and requires histone deacetylase activity.

Hypoxia inducible factor promotes murine allergic airway inflammation and is increased in asthma and rhinitis.

BACKGROUND: New therapies are necessary to address inadequate asthma control in many patients. This study sets out to investigate whether hypoxia-inducible factor (HIF) is essential for development of allergic airway inflammation (AAI) and therefore a potential novel target for asthma treatment. METHODS: Mice conditionally knocked out for HIF-1ß were examined for their ability to mount an allergic inflammatory response in the lung after intratracheal exposure to ovalbumin. The effects of treating wild-type mice with either ethyl-3,4-dihydroxybenzoate (EDHB) or 2-methoxyestradiol (2ME), which upregulate and downregulate HIF, respectively, were determined. HIF-1α levels were also measured in endobronchial biopsies and bronchial fluid of patients with asthma and nasal fluid of patients with rhinitis after challenge. RESULTS: Deletion of HIF-1ß resulted in diminished AAI and diminished production of ovalbumin-specific IgE and IgG(1) . EDHB enhanced the inflammatory response, which was muted upon simultaneous inhibition of vascular endothelial growth factor (VEGF). EDHB and 2ME antagonized each other with regard to their effects on airway inflammation and mucus production. The levels of HIF-1α and VEGF increased in lung tissue and bronchial fluid of patients with asthma and in the nasal fluid of patients with rhinitis after challenge. CONCLUSIONS: Our results support the notion that HIF is directly involved in the development of AAI. Most importantly, we demonstrate for the first time that HIF-1α is increased after challenge in patients with asthma and rhinitis. Therefore, we propose that HIF may be a potential therapeutic target for asthma and possibly for other inflammatory diseases.

Comparison of mibefradil and derivative NNC 55-0396 effects on behavior, cytochrome P450 activity, and tremor in mouse models of essential tremor.

NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2, 3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride], is a mibefradil derivative that retains potent in vitro T-type calcium channel antagonist efficacy. We compared the two compounds for behavioral toxicity, effects on cytochrome P450 activity, and efficacy against tremor in the γ-aminobutyric acid type A (GABAA) receptor subunit α1-null mouse, and the harmaline tremor model of essential tremor in wild-type mice. NNC 55-0396 was better tolerated than mibefradil in the horizontal wire test of sedation/motor function, with 3/6 failing at 300 and 30mg/kg respectively. To assess for a potential interaction with harmaline, mice were given the drugs, followed by harmaline or vehicle, and tested 30min later in the inverted wire grid test. Mibefradil exacerbated, whereas NNC 55-0396 ameliorated harmaline-induced test deficits. In mouse liver microsomes, NNC 55-0396 was a less potent inhibitor of harmaline O-demethylation than mibefradil (Ki: 0.95 and 0.29µM respectively), and also less potent at inhibiting testosterone 6-ß-hydroxylation (Ki: 0.71 and 0.12µM respectively). In the GABAA α1-null model, NNC 55-0396 but not mibefradil, (each at 20mg/kg), suppressed tremor while NNC 55-0396 at 12.5mg/kg suppressed harmaline-induced tremor by half by 20-100min, whereas mibefradil at the same dose did not significantly affect tremor. In contrast to mibefradil, NNC 55-0396 is well tolerated and suppresses tremor, and exerts less cytochrome P450 inhibition. These results suggest potential clinical utility for NNC 55-0396 or similar derivatives as a T-type calcium antagonist.

Human CYP2S1 metabolizes cyclooxygenase- and lipoxygenase-derived eicosanoids.

CYP2S1 is a recently described dioxin-inducible cytochrome P450. We previously demonstrated that human CYP2S1 oxidizes a number of carcinogens but only via the peroxide shunt. In this article, we investigated whether human CYP2S1 can metabolize cyclooxygenase- and lipoxygenase-derived lipid peroxides in a NADPH-independent fashion. Human CYP2S1 metabolizes prostaglandin G(2) (PGG(2)) (K(m) = 0.267 ± 0.072 µM) into several products including 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (12-HHT). It also metabolizes prostaglandin H(2) (PGH(2)) (K(m) = 11.7 ± 2.8 µM) into malondialdehyde, 12-HHT, and thromboxane A(2) (TXA(2)). The turnover to 12-HHT by human CYP2S1 (1.59 ± 0.04 min(-1)) is 40-fold higher than that of TXA(2) (0.04 min(-1)). In addition to PGG(2) and PGH(2) metabolism, human CYP2S1 efficiently metabolizes the hydroperoxyeicosatetraenoic acids (5S-, 12S-, and 15S-) and 13S-hydroperoxyoctadecadienoic acid into 5-oxo-eicosatetraenoic acid (turnover = 16.7 ± 0.3 min(-1)), 12-oxo-eicosatetraenoic acid 1 (11.5 ± 0.9 min(-1)), 15-oxo-eicosatetraenoic acid (16.9 ± 0.8 min(-1)), and 13-octadecadienoic acid (20.2 ± 0.9 min(-1)), respectively. Other cytochromes P450 such as CYP1A1, 1A2, 1B1, and 3A4 underwent similar conversions but at slower rates. The fatty acid hydroperoxides were also converted by human CYP2S1 to several epoxyalcohols. Our data indicate that fatty acid endoperoxides and hydroperoxides represent endogenous substrates of CYP2S1 and suggest that the enzyme CYP2S1 may play an important role in the inflammatory process because some of the products that CYP2S1 produces play important roles in inflammation.