Development of novel CH223191-based antagonists of the aryl hydrocarbon receptor.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates genes involved in drug/xenobiotic metabolism, cell cycle progression, cell fate determination, immune function, and inflammatory response. Increasing evidence that AHR plays a role in the pathophysiology of a number of human disease states is driving the need for improved pharmacological tools to be used for understanding the in vivo impact of AHR modulation. In this study, we have characterized and used structure-activity relationship analyses of a newly synthesized library of derivatives of the potent AHR antagonist 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH223191). Initial screening of these compounds revealed that those bearing groups with strong electronegativity at the R1 position (i.e., CHD-5, CHD-11, and CHD-12) versus those that are more electron-poor at this position (i.e., CHD-7 and CHD-8) elicited the most potent AHR antagonistic properties. The ability of these derivatives to inhibit agonist (2,3,7,8-tetrachlorodibenzo-p-dioxin) binding, nuclear translocation of AHR, and agonist-induced enzyme activity also were determined and support the initial findings. Furthermore, CH223191, but not CHD-5, CHD-11, or CHD-12, was found to exhibit AHR-independent proproliferative properties. These results contribute to our understanding of the structural requirements of potent AHR antagonists and the development of effective pharmacological tools to be used for studying the pathophysiological role of AHR.

Cigarette smoke condensate and dioxin suppress culture shock induced senescence in normal human oral keratinocytes.

The aryl hydrocarbon receptor is a ligand activated transcription factor which regulates biological responses to a variety of environmental pollutants, such as dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) and cigarette smoke. The purpose of this study was to determine whether cigarette smoke condensate (CSC) is capable of activating the AHR in normal human oral keratinocytes (NHOK) and inhibiting their ability to senesce. Towards this end, NHOK were isolated from human subjects and were cultured in the presence or absence of either TCDD or CSC. While neither TCDD nor CSC treatments altered the lifespan of NHOK in culture, both were capable of suppressing a culture induced premature senescence as indicated by their ability to decrease the mRNA and protein levels of the senescence markers p16(INK4a), p53 and p15(INK4b). A role of the AHR in mediating these events is indicated by the observations that the TCDD and CSC-induced decreases in p15(INK4b), p16(INK4a) and p53 expression was accompanied by a corresponding increase in the expression levels of the AHR target gene, CYP1A1. In addition, cotreatment with the AHR antagonist, 3'-methoxy-4'-nitroflavone (MNF) blocked the effects of TCDD and CSC on p53 and CYP1A1 expression. The findings of this study indicate that in NHOK, CSC is capable of altering a key cell fate decision, i.e., commitment to premature senescence, that is in part, dependent on the AHR. These results support the idea that progression of CSC-induced tumorigenesis may include an AHR-mediated inhibition of senescence that contributes to immortalization and agents that block the actions of the AHR may be effective components of novel cancer therapeutics.

Impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cutaneous wound healing.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a representative of a large group of polyhalogenated aromatic hydrocarbons that are widespread environmental contaminants. Administration of TCDD to laboratory animals or cultured cells results in a number of adverse effects that are well documented. For example, the effects of TCDD observed in developing organisms indicate that exposure to this class of environmental contaminants significantly alters embryo morphogenesis. However, it is not clear whether tissue regeneration in adult animals may be similarly affected. With this in mind, we examined the impact of TCDD exposure on wound healing using a murine cutaneous wound healing model. Our results indicate that TCDD exposure did not significantly alter the time needed for wound closure. However, in the TCDD-treated mice, a significant decrease in tensile strength in the healed wounds was observed which is indicative of an aberrantly healed wound. Immunostaining revealed that exposure to TCDD increased the population of macrophages detected within the wounded tissue at the latter stages of wound healing. Our findings support the idea that exposure to environmental contaminants such as TCDD is proinflammatory in the wounded tissue, disrupts normal healing and ultimately produces in a poorly healed wound.

Role of the xenobiotic receptor in inflammatory bowel disease.

BACKGROUND: Gene-environment interplay modulates inflammatory bowel diseases (IBD). Dioxin-like compounds can activate the aryl hydrocarbon receptor (AhR) and alter macrophage function as well as T-cell polarization. We hypothesized that attenuation of the AhR signaling pathway will ameliorate colitis in a murine model of IBD. METHODS: Dextran sulfate sodium (DSS) colitis was induced in C57BL/6 AhR null mice (AhR(-/-) ), heterozygous mice (AhR(-/+) ), and their wildtype (WT) littermates. Clinical and morphopathological parameters were used to compare the groups. PATIENTS: AhR pathway activation was analyzed in biopsy specimens from 25 IBD patients and 15 healthy controls. RESULTS: AhR(-/-) mice died before the end of the treatment. However, AhR(-/+) mice exhibited decreased disease activity compared to WT mice. The AhR(-/+) mice expressed less proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) (6.1- versus 15.7-fold increase) and IL17 (23.7- versus 67.9-fold increase) and increased antiinflammatory IL-10 (2.3-fold increase) compared with the AhR(+/+) mice in the colon. Colonic macrophage infiltration was attenuated in the AhR(-/+) group. AhR and its downstream targets were significantly upregulated in IBD patients versus control (CYP1A1 -19.9, and IL8- 10-fold increase). CONCLUSIONS: Attenuation of the AhR receptor expression resulted in a protective effect during DSS-induced colitis, while the absence of AhR exacerbated the disease. Abnormal AhR pathway activation in the intestinal mucosa of IBD patients may promote chronic inflammation. Modulation of AhR signaling pathway via the diet, cessation of smoking, or administration of AhR antagonists could be viable strategies for the treatment of IBD.

Hereditary persistence of alpha-fetoprotein and H19 expression in liver of BALB/cJ mice is due to a retrovirus insertion in the Zhx2 gene.

The alpha-fetoprotein (AFP) and H19 genes are transcribed at high levels in the mammalian fetal liver but are rapidly repressed postnatally. This repression in the liver is controlled, at least in part, by the Afr1 gene. Afr1 was defined >25 years ago when BALB/cJ mice were found to have 5- to 20-fold higher adult serum AFP levels compared with all other mouse strains; subsequent studies showed that this elevation was due to higher Afp expression in the liver. H19, which has become a model for genomic imprinting, was identified initially in a screen for Afr1-regulated genes. The BALB/cJ allele (Afr1(b)) is recessive to the wild-type allele (Afr1(a)), consistent with the idea that Afr1 functions as a repressor. By high-resolution mapping, we identified a gene that maps to the Afr1 interval on chromosome 15 and encodes a putative zinc fingers and homeoboxes (ZHX) protein. In BALB/cJ mice, this gene contains a murine endogenous retrovirus within its first intron and produces predominantly an aberrant transcript that no longer encodes a functional protein. Liver-specific overexpression of a Zhx2 transgene restores wild-type H19 repression on a BALB/cJ background, confirming that this gene is responsible for hereditary persistence of Afp and H19 in the livers of BALB/cJ mice. Thus we have identified a genetically defined transcription factor that is involved in developmental gene silencing in mammals. We present a model to explain the liver-specific phenotype in BALB/cJ mice, even though Afr1 is a ubiquitously expressed gene.

B-cell and plasma-cell splicing differences: a potential role in regulated immunoglobulin RNA processing.

The immunoglobulin micro pre-mRNA is alternatively processed at its 3' end by competing splice and cleavage-polyadenylation reactions to generate mRNAs encoding the membrane-associated or secreted forms of the IgM protein, respectively. The relative use of the competing processing pathways varies during B-lymphocyte development, and it has been established previously that cleavage-polyadenylation activity is higher in plasma cells, which secrete IgM, than in B cells, which produce membrane-associated IgM. To determine whether RNA-splicing activity varies during B-lymphocyte development to contribute to micro RNA-processing regulation, we first demonstrate that micro pre-mRNA processing is sensitive to artificial changes in the splice environment by coexpressing SR proteins with the micro gene. To explore differences between the splice environments of B cells and plasma cells, we analyzed the splicing patterns from two different chimeric non-Ig genes that can be alternatively spliced but have no competing cleavage-polyadenylation reaction. The ratio of intact exon splicing to cryptic splice site use from one chimeric gene differs between several B-cell and several plasma-cell lines. Also, the amount of spliced RNA is higher in B-cell than plasma-cell lines from a set of genes whose splicing is dependent on a functional exonic splice enhancer. Thus, there is clear difference between the B-cell and plasma-cell splicing environments. We propose that both general cleavage-polyadenylation and general splice activities are modulated during B-lymphocyte development to ensure proper regulation of the alternative micro RNA processing pathways.