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T Thomas

Publications and source records attributed to T Thomas.

At least 37 records · Page 2Linked to original sources

Measurement of TG synthesis and turnover in vivo by 2H2O incorporation into the glycerol moiety and application of MIDA.

A method is presented for measurement of triglyceride (TG) synthesis that can be applied to slow-turnover lipids. The glycerol moiety of TG is labeled from 2H2O, and mass isotopomer distribution analysis (MIDA) is applied. Mice and rats were given 4-8% 2H2O in drinking water; TG-glycerol was isolated from adipose and liver during < or =12-wk of 2H2O labeling. Mass isotopomer abundances in the glycerol moiety of TG were measured by GC-MS. The combinatorial pattern of isotopomers revealed the number of H atoms in glycerol incorporating label from 2H2O (n) to be 3.8-4.0 of a possible 5 for adipose tissue and 4.6-4.8 for liver TG. Hepatic TG-glycerol in fact reached 97% predicted maximal value of label incorporation (4.4-4.6 x body 2H2O enrichment), indicating near-complete replacement of the liver TG pool. Label incorporation into adipose tissue revealed turnover of mesenteric TG to be faster (k = 0.21 day-1) than other depots (k = 0.04-0.06 day-1) in mice. TG isolated from subcutaneous depots of growing adult rats plateaued at 85-90% of calculated maximal values at 12 wk (k = 0.05 day-1), excluding significant dilution by unlabeled alpha-glycerol phosphate. Turnover of plasma TG, modeled from 2H incorporation over 60 min, was 0.06 min-1 (half-life 11.5 min). In summary, use of 2H2O labeling with MIDA of TG-glycerol allows measurement of new alpha-glycerol phosphate-derived TG synthesis and turnover. The hypothesis that mesenteric TG is more lipolytically active than other depots, previously difficult to prove by isotope dilution techniques, was confirmed by this label incorporation approach.

Adipose Tissue↗

Aspirin and diabetes: inhibition of amylin aggregation by nonsteroidal anti-inflammatory drugs.

Islet amyloid polypeptide (amylin), cosecreted with insulin by the pancreatic beta-cells, has an important role in the regulation of islet cell hormone homeoastasis. Deposition of beta-sheet polypeptide fibrils into amyloid deposits is considered to be central to the pathology of a number of amyloidogenic disorders, including type-2 diabetes. Amyloid deposits comprised of beta-sheet fibrillar amylin observed in type-2 diabetics are cytotoxic and may have a prominent role in causing beta-cell dysfunction. The amyloidogenic process may impair beta-cell function before cell death and replacement by amyloid. Preservation of beta-cell viability and insulin secretion is a major objective in diabetic care. Using circular dichroism and Congo red absorption techniques we found that clinically relevant doses of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDS) prevented and also reversed the beta-sheet conformation of human amylin. The specific COX-2 inhibitors were less effective. The anti-inflammatory steroid prednisolone or the analgesic acetaminophen had no effect on amylin fibrillogenesis. This action of NSAIDS was similar to their inhibition of beta-sheet conformation of the Alzheimer protein, amyloid-beta. Aspirin, currently recommended for the prevention of cardiovascular complications in diabetic patients, may also ameliorate the disease process in diabetes by preserving the beta-cell function.

Amyloid↗

[Value of percutaneous core needle biopsy in the investigation of a suspected bone tumor].

PURPOSE: To Determine the value of percutaneous core needle biopsy in the investigation of a suspected bone neoplasm. MATERIAL AND METHODS: We performed a retrospective study of 91 core needle biopsies performed between May 1995 and October 2001. Patients were excluded if they had a known primary carcinoma or if an infection was suspected by clinical, physical or laboratory findings. The results were correlated to the analysis of the surgical piece or, for the 28 patients who did not undergo surgery, to the clinical evolution over more than 12 months. RESULTS: The final diagnosis was metastasis in 29 cases, primary bone tumor in 36 cases and benign lesions in 25 cases. Sensitivity was 92.3% and specificity was 97.4%. For primary malignant bone tumors, results respected histology features and grade in 79.2%. In cases of mistake, because of the correlation of these results to the clinical and radiological context, the treatment of the bony malignant lesions were adapted in 95.8% of cases. Only one major complication was reported in these 91 biopsies. CONCLUSION: First intention core needle biopsy, confronted with radio-clinical context seems to have a place in the evaluation of bone lesions when a tumor is suspected. This technique, less expensive than an open biopsy and with fewer complications, is best performed as part of a multidisciplinary approach with the surgeon's collaboration.

Adult↗

Progestins initiate adverse events of menopausal estrogen therapy.

OBJECTIVE: Recent report, particularly the Women's Health Initiative, demonstrated that hormone therapy with combined estrogen plus progestin increased the incidence of heart attacks, stroke, blood clots, breast cancer and dementia in women over 65 years old. We investigated the role of synthetic progestins in initiating the adverse events associated with estrogen therapy. METHODS: We used a fluorescence imaging technique, which allows video microscopic recordings of blood flow, blood vessel morphology and activities of various blood cells in a live animal. The acute peripheral and cerebrovascular responses were measured following intraperitoneal (5 or 10 mg) or intravenous (10 or 100 microg) administration of progesterone, synthetic progestins (medroxyprogesterone acetate and norethindrone) or estrogens (conjugated equine estrogens and 17 beta-estradiol). RESULTS: In both peripheral and cerebral vasculature, synthetic progestins caused endothelial disruption, accumulation of monocytes in the vessel wall, platelet activation and clot formation, which are early events in atherosclerosis, inflammation and thrombosis. Natural progesterone or estrogens did not show such toxicity. CONCLUSION: The risk associate with combined estrogen plus progestin therapy may be a consequence of vascular actions of progestins. Using progestins with minimal vascular toxicity may lead to safer estrogen preparations for menopausal women.

Animals↗

Effects of aminooxy analogues of biogenic polyamines on aggregation and stability of calf thymus DNA.

The effect of a series of aminooxy analogues of the biogenic polyamines spermidine and spermine on the conformation of calf thymus DNA is studied. These new molecules are isosteric and charge insufficient analogues that are suitable to study the roles of both charge distribution and structural requirements in the molecular physiology of the biogenic polyamines. They are also evidenced as useful tools to inhibit polyamine biosynthesis and cell growth. Circular dichroism (CD) spectra of solutions containing DNA and the aminooxy analogues at different concentrations (100-1000 microM) and different pH values, (5-7.5) are recorded. We use both sonicated and highly polymerized calf thymus DNA. The CD spectra of sonicated DNA showed the formation of Psi-DNA, a highly ordered aggregated structure similar to liquid crystals, in the presence of the aminooxy analogues. Aggregation induced by an aminooxy derivative of spermine is followed by DNA collapse when increasing the polyamine concentration. The features of Psi-DNA are not detected for highly polymerized DNA. Temperature melting measurements support a high degree of structural order of the aggregates. The CD experiments indicate that dications are unable to induce major changes on the macromolecular structure of DNA. In addition, aggregation is only observed when the trimethylene moiety is present between two adjacent positive charges. The observed differences among the CD spectra of DNA solutions with different aminooxy derivatives of spermidine indicate different roles for different amino groups of this biogenic polyamine when interacting with DNA. Our results support the idea that aminooxy analogues can be used as good models in studying the physiological functions of biogenic polyamines.

Animals↗

Overexpression of White Collar-1 (WC-1) activates circadian clock-associated genes, but is not sufficient to induce most light-regulated gene expression in Neurospora crassa.

Many processes in fungi are regulated by light, but the molecular mechanisms are not well understood. The White Collar-1 (WC-1) protein is required for all known blue-light responses in Neurospora crassa. In response to light, WC-1 levels increase, and the protein is transiently phosphorylated. To test the hypothesis that the increase in WC-1 levels after light treatment is sufficient to activate light-regulated gene expression, we used microarrays to identify genes that respond to light treatment. We then overexpressed WC-1 in dark-grown tissue and used the microarrays to identify genes regulated by an increase in WC-1 levels. We found that 3% of the genes were responsive to light, whereas 7% of the genes were responsive to WC-1 overexpression in the dark. However, only four out of 22 light-induced genes were also induced by WC-1 overexpression, demonstrating that changes in the levels of WC-1 are not sufficient to activate all light-responsive genes. The WC proteins are also required for circadian rhythms in dark-grown cultures and for light entrainment of the circadian clock, and WC-1 protein levels show a circadian rhythm in the dark. We found that representative samples of the mRNAs induced by over-expression of WC-1 show circadian fluctuations in their levels. These data suggest that WC-1 can mediate both light and circadian responses, with an increase in WC-1 levels affecting circadian clock-responsive gene regulation and other features of WC-1, possibly its phosphorylation, affecting light-responsive gene regulation.

Circadian Rhythm↗

CML leukapheresis products can be enriched for CD34+ cells and simultaneously depleted of CD15+ cells using a simple Ab cocktail.

BACKGROUND: CML progenitor-cell studies would be greatly facilitated if samples could be repeatedly accessed from a source of well-characterized cells. The present study was designed to develop a simple, inexpensive Ab cocktail that would provide subpopulations of cells enriched for CD34+ cells and simultaneously depleted of CD15+ mature myeloid cells. METHODS: Cells from leukapheresis products from CML patients at diagnosis were incubated with each of two Ab cocktails. The standard cocktail (debulking, DB), containing 11 Abs, is recommended for obtaining a highly enriched population of CD34+ cells. The efficacy of an alternative, simpler cocktail (CML custom, CC), containing only four Abs was tested. The recoveries of CD34+ cells, CD15+ cells, colony-forming unit granulocyte-macrophage, and LTCIC were monitored. The samples were then cryopreserved, thawed, and the recoveries remeasured. RESULTS: The purity of CD34+ cells was significantly superior using the DB cocktail than with the CC cocktail. Conversely, using the CC cocktail, the yield of CD34+ cells was significantly higher compared to the DB cocktail. These results were maintained even when the amount of Ab was reduced 10-fold. Both Ab cocktails consistently removed > 99% of the CD15+ cells. Consistent with the CD34+ cell-enrichment data, higher colony-forming cell (CFC) frequencies were obtained with the DB cocktail, although superior yields of CFC were obtained with the CC cocktail. After cryopreservation and thawing the yield of CD34+ cells remained high, and a further reduction in the number of CD15+ cells was obtained. DISCUSSION: A method is described that allows the rapid and efficient debulking of large CML samples. This strategy will provide a source of well-characterized CML stem/progenitor cells that can be repeatedly accessed.

Antigens, CD34↗

Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension.

Accumulating evidence suggests that stellate cells are involved in the regulation of the liver microcirculation and portal hypertension. Activated hepatic stellate cells have the necessary machinery to contract or relax in response to a number of vasoactive substances. Because stellate cells play a role in both fibrosis and portal hypertension, they are currently regarded as therapeutic targets to prevent and treat the complications of chronic liver disease.

Cell Communication↗

Synchrotron radiation microtomography allows the analysis of three-dimensional microarchitecture and degree of mineralization of human iliac crest biopsy specimens: effects of etidronate treatment.

Quantitative microcomputed tomography using synchrotron radiation (SR microCT) was used to assess the effects of a sequential etidronate therapy on both three-dimensional (3D) microarchitecture and degree of mineralization of bone (DMB) in postmenopausal osteoporosis. Thirty-two iliac crest biopsy specimens were taken from 14 patients with osteoporosis (aged 64 +/- 1.8 years) before (baseline) and after 1 year of etidronate treatment, and after 2 years of treatment for four of the patients. The samples were imaged at high spatial resolution (voxel size = 10 microm) using the microtomography system developed at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. Three-dimensional microarchitecture parameters were calculated and compared with those obtained from conventional histomorphometry. In addition, the DMB was evaluated also in 3D. No significant statistical changes regarding bone mass and structural parameters were observed in histomorphometry or 3D analyses. The distribution of the DMB in cortical and trabecular bone showed a trend to a shift toward highest mineralization values after 1 year of etidronate treatment (3.88% and 1.24% in cortical and trabecular bone, respectively). This trend was more evident after 2 years. The study also showed that SR microCT is an accurate technique and the only one for quantifying both the mineralization and the microarchitecture of bone samples at the same time in 3D.

Aged↗

DNA condensation by polyamines: a laser light scattering study of structural effects.

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.

Bacteriophage lambda↗

Aspirin and non-steroidal anti-inflammatory drugs inhibit amyloid-beta aggregation.

The neurotoxic and proinflammatory actions of the Alzheimer peptide amyloid-beta (Abeta) are dependent on its aggregation and beta-sheet conformation. Chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin for arthritis decreases the risk of developing Alzheimer's disease (AD) by unknown mechanisms. We report that these drugs inhibit human Abeta aggregation in vitro and reverse the beta-sheet conformation of preformed fibrils at clinically relevant doses. Aspirin prevented enhanced Abeta aggregation by aluminum, an environmental risk factor for AD. This anti-aggregatory effect was restricted to NSAIDs and was not exhibited by other drugs used in AD therapy. NSAIDS may have a role in the prevention and treatment of AD, in addition to a number of age-related disorders such as arthritis, cardiovascular disease and cancer.

Alzheimer Disease↗

Purification of a pluripotent neural stem cell from the adult mouse brain.

The adult mammalian central nervous system (CNS) contains a population of neural stem cells (NSCs) with properties said to include the generation of non-neural progeny. However, the precise identity, location and potential of the NSC in situ remain unclear. We purified NSCs from the adult mouse brain by flow cytometry, and directly examined the cells' properties. Here we show that one type of NSC, which expresses the protein nestin but only low levels of PNA-binding and HSA proteins, is found in both ependymal and subventricular zones and accounts for about 63% of the total NSC activity. Furthermore, the selective depletion of the population of this stem cell in querkopf mutant mice (which are deficient in production of olfactory neurons) suggests that it acts as a major functional stem cell in vivo. Most freshly isolated NSCs, when co-cultured with a muscle cell line, rapidly differentiated in vitro into myocytes that contain myosin heavy chain (MyHC). This demonstrates that a predominant, functional type of stem cell exists in the periventricular region of the adult brain with the intrinsic ability to generate neural and non-neural cells.

Animals↗

A molecular beacon strategy for the thermodynamic characterization of triplex DNA: triplex formation at the promoter region of cyclin D1.

We studied the formation of triplex DNA in the purine-pyrimidine-rich promoter site sequence of cyclin D1, located at -116 to -99 from the transcription initiation site, with a molecular beacon comprised of a G-rich 18-mer triplex forming oligodeoxyribonucleotide. Formation of triplex DNA was monitored by enhanced fluorescence of the beacon, due to the weakening of fluorescence energy transfer, upon its binding to the target duplex. Electrophoretic mobility shift assay confirmed triplex DNA formation by these oligonucleotides. In low salt buffer (10 mM Na(+)), triplex DNA formation was not observed in the absence of a ligand such as spermine. At room temperature (22 degrees C), the equilibrium association constant (K(a)) calculated in the presence of 1 microM spermine and 10 mM Na(+) was 3.2 x 10(8) M(-1). The K(a) value was 1.0 x 10(9) M(-1) in the presence of 150 mM Na(+), and it increased by 10-fold by the addition of 1 mM spermine. Delta H, Delta S, and Delta G of triplex DNA formation, calculated from the temperature dependence of K(a) in the range of 20--45 degrees C, were -35.9 kcal/mol, -77 cal/(mol.K), and -13 kcal/mol, respectively, in the presence of 150 mM NaCl. The corresponding values were -52.9 kcal/mol, -132.5 cal/(mol.K), and -13.4 kcal/mol in the presence of 150 mM NaCl and 1 mM spermine. Structurally related polyamines exerted different degrees of triplex DNA stabilization, as determined by binding constant measurements. Comparison of spermine versus hexamine showed a 17-fold increase in the equilibrium association constant, whereas bis(ethyl) derivatization lead to a 4-fold decrease of this value. In the absence of added duplex and polyamines, the molecular beacon dissociated with a melting temperature of 67 degrees C. Thermodynamic parameters of beacon melting were calculated from the melting curve, and the Delta H, Delta S, and Delta G values were 37.8 kcal/mol, 112 cal/(mol.K), and 4.4 kcal/mol, respectively. These results demonstrate that molecular beacons can be used for the direct determination of the equilibrium association constants and thermodynamic parameters of triplex DNA formation in the presence of ligands such as polyamines.

Cyclin D1↗

Increase in the stability and helical content of estrogen receptor alpha in the presence of the estrogen response element: analysis by circular dichroism spectroscopy.

Ligand-dependent stabilization of the estrogen receptor (ER) is often postulated, with limited support from experimental data. We studied the thermal unfolding of recombinant ERalpha by circular dichroism (CD) spectroscopy. The T(M) of unfolding of ERalpha was 38 +/- 2.4 degrees C, and the van't Hoff enthalpy of unfolding was 31.7 +/- 3.4 kcal/mol in the absence of ligands. Addition of estradiol (E(2)) increased the T(M) to 43.6 +/- 2.3 degrees C, while addition of E(2) and an oligonucleotide harboring the estrogen response element (ERE) increased the T(M) to 47.9 +/- 1.6 degrees C. Addition of the antiestrogen 4-hydroxytamoxifen (HT) alone did not increase the T(M); however, a combination of HT and the ERE increased the T(M) to 48.9 +/- 1.0 degrees C. The ERE alone increased the T(M) to 46.1 +/- 0.9 degrees C. Addition of E(2) alone had no effect on the apparent enthalpy of unfolding; however, the ERE alone increased the apparent enthalpy from 31.7 to 36.1 kcal/mol. ERalpha samples containing the ERE also exhibited an increase in the negative ellipticity at 208 and 222 nm, relative to that of ligand-free ERalpha, suggesting a stabilization of the alpha-helix. CD data analysis further showed that the presence of the ERE caused a large increase in alpha-helical content of ERalpha in both the presence and absence of the ligands. This increase in alpha-helical content of ERalpha was not observed in the presence of a nonspecific oligonucleotide. These results show that the ERE can increase the thermal stability of ERalpha, enhance its alpha-helical content, and facilitate the cooperativity of the folding transition.

Circular Dichroism↗

Regulation of estrogenic and nuclear factor kappa B functions by polyamines and their role in polyamine analog-induced apoptosis of breast cancer cells.

The natural polyamines -putrescine, spermidine, and spermine- are essential for cell growth and differentiation. Polyamines are involved in several gene regulatory functions, although their mechanism(s) of action has not been elucidated. We investigated the role of polyamines in the function of NF-kappa B and estrogen receptor-alpha (ER alpha), two transcription factors implicated in breast cancer cell proliferation and cell survival, using MCF-7 breast cancer cells. We found that spermine facilitated the binding of ER alpha and NF-kappa B to estrogen response element (ERE)- and NF-kappa B response element (NRE), respectively, and enhanced ER alpha-mediated transcriptional activation in transient transfection experiments. We also found that the association of the co-regulatory protein CBP/p300 with ER alpha and NF-kappa B was increased by spermine treatment of MCF-7 cells. Spermine also increased the nuclear translocation of NF-kappa B compared to the control. In contrast, treatment of MCF-7 cells with polyamine analogs, BE-3-4-3 and BE-3-3-3, resulted in transcriptional inhibition of both ERE- and NRE-driven reporter plasmids. In addition, polyamine analogs inhibited the association of ER alpha and NF-kappa B with CBP/p300 and were unable to facilitate nuclear translocation of NF-kappa B. APO-BRDU assay demonstrated that polyamine analogs induced apoptosis, with a loss of the anti-apoptotic protein Bcl-2. These data show a gene regulatory function of polyamines involving transcriptional activation of ER alpha and NF-kappa B, potentially leading to the up-regulation of genes involved in breast cancer cell proliferation. Our results with BE-3-4-3 and BE-3-3-3 suggest that down-regulation of ER alpha- and NF-kappa B-regulated genes is a possible mechanism for the action of polyamine analogs in inducing apoptosis of breast cancer cells.

Apoptosis↗

Comparative analysis and application of fluorescent protein-tagged connexins.

In order to examine connexin transport, assembly, and turnover in living cells, we tagged green fluorescent protein or its color variants to several members of the connexin family of proteins. When green fluorescent protein was tagged to the carboxyl terminal end of connexin43 (Cx43-GFP), the resulting fusion protein was transported and assembled into functional gap junctions. However, when GFP was tagged to the amino terminal end of Cx43 (GFP-Cx43), this chimera was biosynthesized, transported to the plasma membrane, but failed to form gap junction channels that could transfer Lucifer yellow. Single cells that expressed Cx43-GFP were capable of transporting this fusion protein to the cell surface in the absence of cell-cell contact. Imaging of Cx43-yellow (Y)FP (Cx43-YFP) was quite efficient; however, the low quantum yield Cx43-BFP and the requirement for ultraviolet excitation made this chimera less suitable for time-lapse imaging. Cx43-cyan C(FP) (Cx43-CFP) was more suitable for imaging than Cx43-blue (B)FP and could be effectively separated from Cx43-YFP. The versatility of tagging GFP to the carboxyl terminal end of other members of the connexin family was established when Cx32-GFP and Cx26-YFP were found to assemble into gap junctions capable of transferring Lucifer yellow. Finally, we are examining the effectiveness of using a new red fluorescent protein (DsRed) fused to connexins in combination with Cx-GFP to simultaneously examine the kinetics, transport and turnover of two connexins. Together, our studies suggest that tagging fluorescent proteins to the carboxyl terminal end of connexins is an effective and valuable approach for studying the life cycle and dynamics of connexins in living cells.

Animals↗

Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7 breast cancer cells using suppression subtractive hybridization.

Aryl hydrocarbon receptor (AhR) agonists inhibit 17beta-estradiol (E2) induced growth of MCF-7 human breast cancer cells in vitro and rodent mammary tumor growth in vivo. Genes associated with inhibitory AhR-estrogen receptor (ER) crosstalk were investigated in MCF-7 human breast cancer cells using poly(A)(+)RNA from cells treated with either 1 nM E2 (target) or E2 plus 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (reference) or 25 microM diindolylmethane (DIM) as AhR agonists in MCF-7 cells. Suppression subtractive hybridization (SSH) was subsequently used to identify 33 genes with sequence homology to known human genes that are induced by E2 and inhibited by AhR agonists in MCF-7 cells; two unknown genes were also identified. Many of these genes are involved in cell proliferation and these include cell cycle regulators (cdc28/cdc2-associated protein), nucleotide synthases (thymidylate synthase), early intermediate genes (early growth response alpha, EGRalpha) and other proteins involved in signaling pathways (calmodulin, ATP synthase alpha subunit). Thus SSH has identified a diverse spectrum of new genes that are affected by inhibitory AhR-ER crosstalk and among this group are a subset of genes that may be critical for the in vivo antitumorigenic effects of AhR agonists.

Animals↗