Thomas A. Gasiewicz

Role of the MEOX2 homeobox gene in neurovascular dysfunction in Alzheimer disease.

Neurovascular dysfunction substantially contributes to Alzheimer disease. Here, we show that transcriptional profiling of human brain endothelial cells (BECs) defines a subset of genes whose expression is age-independent but is considerably altered in Alzheimer disease, including the homeobox gene MEOX2 (also known as GAX), a regulator of vascular differentiation, whose expression is low in Alzheimer disease. By using viral-mediated MEOX2 gene silencing and transfer, we show that restoring expression of the protein it encodes, GAX, in BECs from individuals with Alzheimer disease stimulates angiogenesis, transcriptionally suppresses AFX1 forkhead transcription factor–mediated apoptosis and increases the levels of a major amyloid-β peptide (Aβ) clearance receptor, the low-density lipoprotein receptor–related protein 1 (LRP), at the blood-brain barrier. In mice, deletion of Meox2 (also known as Gax) results in reductions in brain capillary density and resting cerebral blood flow, loss of the angiogenic response to hypoxia in the brain and an impaired Aβ efflux from brain caused by reduced LRP levels. The link of MEOX2 to neurovascular dysfunction in Alzheimer disease provides new mechanistic and therapeutic insights into this illness.

2,3,7,8-Tetrachlorodibenzo-p-dioxin tissue distribution, excretion, and effects on clinical chemical parameters in guinea pigs

The tissue distribution of 14C-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in adult male guines pigs was studied up to 15 days following its ip injection (2.0 μg/kg). On Day 1, the highest levels of radioactivity (% of original dose/g tissue) were located in the adipose tissue, adrenals, liver, spleen, intestine, and skin. All other tissues examined contained less than 0.3%/g tissue. By Day 15, the level of radioactivity in the liver increased to nearly three times its initial value. An increase in radioactivity was also noted in the adrenals, kidneys, and lungs. These increases appeared to be due to the mobilization of fat stores and the subsequent redistribution of radioactivity contained in these stores to other organs. Following a single intraperitoneal dose of 0.5 μg [3H]TCDD/kg the excretion of 3H in the urine and feces appeared to be linear up to 23 days. Assuming the excretion of radioactivity would continue in a linear manner, the time for excretion of half the administered dose by way of the urine and feces was calculated to be 30.2 ± 5.8 days. The effect of TCDD (1.0 μg/kg) upon various clinical chemical parameters was determined periodically up to 14 days and compared to pairfed controls. Statistically significant increases in plasma albumin, total protein, iron, urea nitrogen, cholesterol, and triglycerides were observed in TCDD-treated pigs.

Novel Compound 2-Methyl-2H-pyrazole-3-carboxylic Acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191) Prevents 2,3,7,8-TCDD-Induced Toxicity by Antagonizing the Aryl Hydrocarbon Receptor

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant with many toxic effects, including endocrine disruption, reproductive dysfunction, immunotoxicity, liver damage, and cancer. These are mediated by TCDD binding to and activating the aryl hydrocarbon receptor (AhR), a basic helix-loop-helix transcription factor. In this regard, targeting the AhR using novel small molecule inhibitors is an attractive strategy for the development of potential preventive agents. In this study, by screening a chemical library composed of approximately 10,000 compounds, we identified a novel compound, 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191), that potently inhibits TCDD-induced AhR-dependent transcription. In addition, CH-223191 blocked the binding of TCDD to AhR and inhibited TCDD-mediated nuclear translocation and DNA binding of AhR. These inhibitory effects of CH-223191 prevented the expression of cytochrome P450 enzymes, target genes of the AhR. Unlike many known antagonists of AhR, CH-223191 did not have detectable AhR agonist-like activity or estrogenic potency, suggesting that CH-223191 is a specific antagonist of AhR. It is noteworthy that CH-223191 potently prevented TCDD-elicited cytochrome P450 induction, liver toxicity, and wasting syndrome in mice. Taken together, these results demonstrate that this novel compound, CH-223191, may be a useful agent for the study of AhR-mediated signal transduction and the prevention of TCDD-associated pathology.

The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxylapatite.

Abstract An assay using hydroxylapatite has been developed for the examination and quantitation of cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This method, as compared to others, has a relatively high ratio of specific to nonspecific binding of [3H]TCDD and is relatively rapid. The total number of specific binding sites and equilibrium dissociation constants (KD) for [3H]TCDD were determined in hepatic cytosol from Sprague-Dawley rats and C57BL 6J , DBA 2J , and B6D2F 1 J mice. With the exception of the cytosol from DBA 2J mice in which little specific binding was observed, high-affinity, specific binding was obtained in all cases. The rat hepatic cytosol demonstrated the highest affinity for [3H]TCDD with a KD of 0.12 n m . The total amount of specific [3H]TCDD binding in hepatic cytosol from B6D2F 1 J mice was found to be intermediate between that of the C57BL 6J and DBA 2J parents. Ligand competition studies suggest the specificity of binding of various compounds to the receptor species from Sprague-Dawley rats is similar to that observed with the hepatic cytosol from C57BL 6J mice. These data support the hypothesis that, as in mice, the particular cytosol binding species in rats is the receptor that may mediate the induction of aryl hydrocarbon hydroxylase activity.

Analysis of Structural Requirements for Ah Receptor Antagonist Activity: Ellipticines, Flavones, and Related Compounds

A number of studies have examined the structure-activity relationships for the agonist activity of Ah receptor (AhR) ligands. Fewer studies have considered the structural basis for potential antagonist properties. Certain ellipticine derivatives have been reported to bind to the AhR and inhibit the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to transform the AhR to a form that recognizes a dioxin-responsive enhancer element (DRE) upstream of the cytochrome P4501A1 gene. In the present study, over 30 ellipticine derivatives and structurally related compounds were examined for their ability to bind to the AhR, activate it to a DRE-binding form, induce the luciferase gene under control of a DRE-containing enhancer, and block activation of the AhR by TCDD. The ability of several ellipticine derivatives to inhibit TCDD-elicited DRE binding and TCDD-induced luciferase activity was inversely related to their ability to alone stimulate these responses. The most potent antagonist activity was related to good AhR binding characteristics in terms of conforming to previously predicted 14 x 12 x 5 A van der Waals dimensions and the presence of an electron-rich ring nitrogen at or near a relatively unsubstituted X-axis terminal position. Based on these data, a number of flavone derivatives were synthesized and tested for their relative agonist/antagonist activity. These additional data were consistent with the hypothesis that an electron-rich center near or along a lateral position of the van der Waals binding cavity is a characteristic that enhances AhR antagonist activity.

Tissue distribution, excretion, and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the Golden Syrian hamster

The hamster has been reported to be the least sensitive mammalian species to the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The fate of a single dose of [3H]- or [14C]TCDD (650 μg/kg, ip or po) was assessed in male hamsters for up to 35 days following treatment. The greatest content (percentage dose/g tissue) of radioactivity was found in the liver, adipose tissue, and adrenals. The radioactivity in liver and adipose tissue was identified as unmetabolized TCDD. The rate of 3H or 14C elimination in urine and feces suggested a first-order process. Similar half-life of elimination (t12) values of 12.0 ± 2.0 and 10.8 ± 2.4 days (mean ± SD) were obtianed with ip administered [3H]- and [14C]TCDD, respectively. With both [3H]- and [14C]TCDD, approximately 35 and 50% of the radioactivity was eliminated in urine and feces, respectively. The t12 for po administered [3H]TCDD was 15.0 ± 2.5 days. High-pressure liquid chromatography of the urine and bile of animals receiving [14C]TCDD revealed one major and several minor radioactive peaks, none of which corresponded to [14C]TCDD. The apparent absence of TCDD metabolites in extracts of liver or adipose tissue indicates that the biotransformed products of TCDD are readily excreted in urine and bile. The enhanced rate of metabolism and excretion of TCDD in hamsters relative to other species may in part contribute to, but not totally explain its unusual resistance to TCDD toxicity.

The Aryl Hydrocarbon Receptor Attenuates Tobacco Smoke-induced Cyclooxygenase-2 and Prostaglandin Production in Lung Fibroblasts through Regulation of the NF-κB Family Member RelB

Diseases such as chronic obstructive pulmonary disease and lung cancer caused by cigarette smoke affect millions of people worldwide. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that influences responses to certain environmental pollutants such as tobacco smoke. However, the physiological function(s) of the AhR is unknown. Herein we propose that the physiologic role of the AhR is to limit inflammation. We show that lung fibroblasts from AhR–/– mice produce a heightened inflammatory response to cigarette smoke, typified by increased levels of cyclooxygenase-2 (COX-2) and prostaglandins (PGs), when compared with wild type (AhR+/+) fibroblasts. This response was dependent on AhR expression as transient transfection of an AhR expression plasmid into AhR–/– fibroblasts significantly attenuated the smoke-induced COX-2 and PG production, confirming the anti-inflammatory role of the AhR. The AhR can interact with NF-κB. However, the heightened inflammatory response observed in AhR–/– fibroblasts was not the result of NF-κB (p50/p65) activation. Instead it was coupled with a loss of the NF-κB family member RelB in AhR–/– fibroblasts. Taken together, these studies provide compelling evidence that AhR expression limits proinflammatory COX-2 and PG production by maintaining RelB expression. The association between RelB and AhR may represent a new therapeutic and more selective target with which to combat inflammation-associated diseases.

(-)-Epigallocatechin-3-gallate is a novel Hsp90 inhibitor.

(-)-Epigallocatechin-3-gallate (EGCG), a major component of green tea, protects against certain types of cancers, although the mechanism has not yet been determined. It was previously demonstrated that EGCG blocks aryl hydrocarbon receptor (AhR)-mediated transcription induced by the potent carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Unlike other AhR antagonists that directly bind to the AhR, EGCG inhibits AhR-mediated transcription by binding to hsp90. We hypothesize that EGCG exerts anti-AhR and anticancer effects by acting as an hsp90 inhibitor. Using proteolytic footprinting, immunoprecipitation, and an ATP-agarose pull-down assay, EGCG was found to directly modulate the conformation of hsp90 and bind at or near to a C-terminal ATP binding site. Hsp90 chaperone function, as assessed by its ability to mediate refolding of denatured luciferase, was inhibited by EGCG treatment. Hsp90 dimerization, which occurs at the C-terminal end, was also inhibited by EGCG treatment. Coimmunoprecipitation studies showed that EGCG stabilizes an AhR complex that includes hsp90 and XAP2 (hepatitis B virus X-associated protein 2), and decreases the association of aryl hydrocarbon nuclear translocator (Arnt) with ligand-activated AhR. Thus, EGCG, through its ability to bind to hsp90, blocks AhR response element (AhRE) recognition. These studies indicate a novel mechanism whereby EGCG inhibits ligand-induced AhRE binding and AhR-mediated transcriptional activity. In EGCG-treated human ovarian carcinoma SKOV3 cells, decreased levels of several cancer-related hsp90 client proteins, such as ErbB2, Raf-1 and phospho-AKT, were observed. EGCG also modified the association of hsp90 with several cochaperones. Overall, these data indicate that EGCG is a novel hsp90 inhibitor. Further studies are needed to determine if this has a role in the antitumor actions of EGCG.

Role of Estrogen Receptor α in Hematopoietic Stem Cell Development and B Lymphocyte Maturation in the Male Mouse1

Although estrogens and estrogen receptors (ERs) are known to function in the male brain and reproductive tract, few studies have evaluated their involvement in the male hematopoietic and immune systems. This study was undertaken to determine the role of ERα in hematopoietic progenitor and B lymphocyte maturation. ERα knockout (ER−/−), wild-type (ER+/+), and radiation chimeric (ERα positive or negative in either nonhematopoietic or hematopoietic elements, or both) male mice were used to determine target tissues. ER−/− and ER+/+ animals showed similar hematopoietic progenitor profiles, but the ER−/− animals had fewer cells in all bone marrow B lymphocyte subpopulations. Animals receiving a pharmacological dose (5 mg/kg BW) of 17β-estradiol (E2) with both elements, ER+/+, had decreased early hematopoietic progenitors and a shift toward a mature B cell subpopulation, whereas animals with both elements, ER−/−, showed changes only in early hematopoietic progenitors. Hematopoietic element ER+/+ animals exhibited...

Studies of the mechanisms of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

The major interest of our laboratory relative to TCDD has been the biochemical mechanism or mechanisms of acute lethality of this compound. As has been noted by other speakers a t this conference, the major feature of a single lethal dose of TCDD across all animal species is a progressive weight loss and general debilitation of the animal with death occurring in a few weeks or a few months. Gross and histopathology have not, to date, provided information that has been particularly useful in focusing biochemical investigations. For example thymic involution is a consistent finding in all animal species. However, numerous investigations have yet to provide information as to the mechanism of thymic involution. Also the major pathologic effect of TCDD in mice and rats is degeneration of the liver. However, the degree of injury to the liver does not appear to be sufficient to be the cause of death. In addition, in the guinea pig, the species most sensitive to TCDD, little damage to the liver is seen. The extreme lethality of TCDD, particularly i n the guinea pig, suggests that TCDD is probably affecting some fundamental process in animal cells leading to a general disfunction of those cells. Studies of the biochemical effects of TCDD intoxication have focused on some of these fundamental processes. Thus, Greig and coworkers’ have shown that liver DNA synthesis stimulated by partial hepatectomy of rats does not appear to be inhibited by TCDD. Also Lucier et d2 have shown that state 3 and state 4 respiration of liver mitochondria, the process by which ATP is synthesized, and ADP:O are not affected in TCDD-treated rats. In our laboratory we have also examined state 3 and state 4 respiration of liver mitochondria from terminally i l l rats treated with TCDD and find state 3 and state 4 respiration is progressing normally. Work by Cunningham and Williams’ indicates that protein and lipid synthesis also appear to be continuing normally in TCDD-treated rats. Work by Vos el ~ 1 . ~ has indicated that TCDD has a marked effect on the immunocompetence of young rodents. However, work by Grieg el al.’ has shown that specific pathogenfree and germ-free rats are as susceptible to the lethal effects of TCDD as normal rats. Thus the reduced immunocompetence is not likely the cause of the acute lethality of TCDD. We have carried out a number of studies examining for effects of TCDD on other fundamental biochemical processes within mammalian cells. It must be stated at the outset that these and other studies have not yet provided us with information on which to base a logical hypothesis for the mechanism or mechanisms of the acute lethal effects of TCDD. However, the data that have been forthcoming from the various studies, in conjunction with data from future experiments, may eventually allow investigators in this field to postulate a mechanism for the acute lethal effects of TCDD. One of the initial studies in our laboratory was based on an article by Jackson’ who reported that TCDD at a concentration of 0.1 pg/liter in the culture medium caused a significant inhibition of all phases of mitosis of dividing endosperm cells of the African