ABSTRACT
We studied effects of nutrient quercetin on cytochromes' Ð 450 1Ð (CYP1A) activities (measured spectrofluorimetrically using 7-ethoxy-resorufin for CYP1A1 and 7-methoxy-resorufin for CYP1A2 as substrates), on mRNA levels (measured by RT-PCR), and on DNA-binding activities (evaluated by an electrophoretic mobility shift assay) of proteins regulating CYP1A expression in untreated and benzo(α)pyrene (BaP)-treated rats. Wistar rats received quercetin, BaP, or both once daily for 1-3 days. Quercetin did not influence CYP1A1 in untreated rats but inhibited BaP-mediated CYP1A induction on the transcriptional level decreasing positive input (AhR functional activity) and increasing negative input (AhRR/ARNT expression and Oct-1 and C/EBP functional activities).
Subject(s)
Antioxidants/pharmacology , Aryl Hydrocarbon Hydroxylases/genetics , Benzo(a)pyrene/toxicity , Carcinogens/toxicity , Mutagens/toxicity , Quercetin/pharmacology , Animals , Environmental Pollutants/toxicity , Gene Expression Regulation , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, WistarABSTRACT
It is known that the metabolic syndrome (MS), which includes hypertension, dislipidemia, glucose intolerance, and obesity leads to cardiovascular diseases. The MS risk is growing catastrophically. Molecular mechanisms allowing to understand the reason of integrated dysfunctions, taking place at MS cases, have remained almost unstudied. The chronical stress plays a crucial role in MS development; therefore in the present work a hypertensive rat strain with Inherited Stress-Induced Arterial Hypertension (ISIAH) was used as a model. It was shown that ISIAH rat strain as compared with the control WAG rat strain is characterized by increased content of triglyceride, VLDL and LDL cholesterols, a decreased content of HDL cholesterol, a high level of apolipoprotein B-100, and decreased level of apolipoprotein A-I. The ISIAH rats body weight was higher as compared with WAG rats; ISIAH rats blood glucose content was higher too. Thus, strain hypertension for ISIAH rat is accompanied by dislipidemia, increased glucose content, and increased body weight, representing a whole set of MS signs. Since at MS cases the systemic abnormalities in lipid and carbohydrate metabolism take place, the functional activity of transcription factors (TFs) participating in integral regulation of lipid and carbohydrate metabolism genes in liver was measured. PPAR, LXR, PXR, CAR DNA-binding activity was increased in ISIAH rats, suggesting involvement of these TFs in MS development. Integrated investigation of PPAR, LXR, PXR, CAR regulatory mechanisms, signal transduction and transcriptional targets will provide insights into the pathogenesis of MS and offer valuable information for designing of drugs for MS treatment.
Subject(s)
Hypertension/metabolism , Liver/metabolism , Metabolic Syndrome/metabolism , Peroxisome Proliferator-Activated Receptors/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/metabolism , Animals , Blood Glucose/metabolism , Body Weight , Carbohydrate Metabolism , Constitutive Androstane Receptor , Dyslipidemias/metabolism , Dyslipidemias/pathology , Hypertension/pathology , Lipid Metabolism , Lipoproteins/biosynthesis , Liver/pathology , Male , Metabolic Syndrome/pathology , Pregnane X Receptor , Rats , Signal Transduction , Transcription, GeneticABSTRACT
Earlier it was shown that male mice of the DD/He strain were highly susceptible to ortho-aminoasotoluene (OAT) induced hepatocarcinogenesis, and resistant to spontaneous liver tumor development as compared to the CC57BR/Mv strain. In the present work we have made a comparative investigation of peroxisome proliferator-activated receptor (PPAR), liver X-receptor (LXR) and retinoic X-receptor (RXR) mRNA levels in liver as well as concentrations of corticosterone, glucose, lipids and insulin in blood of male DD/He and CC57BR/Mv mice. Using the multiplex RT-PCR method it was found that PPAR-alpha, PPAR-gamma, RXR-alpha and RXR-beta mRNA content was essentially decreased in the liver of DD mice as compared to mice of the CC57BR strain. No significant interstrain differences of LXR-alpha and LXR-beta mRNA content were found. In DD micetere was more then the 3-fold decrease of blood content of corticosterone, which is involved in PPAR and RXR regulation. DD mice demonstrated a significant decrease in blood serum glucose and insulin concentrations as well as higher reactivity to insulin as compared with CC57BR mice. Elevated blood total cholesterol and cholesterol HDL level were found in DD mice whereas triglyceride content was basically the same in both mouse strains. It is known that glucocorticoids, PPAR and RXR play crucial role in transcription regulation of inflammation response. Therefore our data allow to suggest that decreased corticosterone level in blood, PPAR and RXR mRNA content in liver of the DD strain may lead to induction of inflammation by OAT exposure, resulting in a high incidence of tumorigenesis in this strain.
Subject(s)
Blood Glucose/metabolism , Lipids/blood , Liver Neoplasms/metabolism , Orphan Nuclear Receptors/biosynthesis , Peroxisome Proliferator-Activated Receptors/biosynthesis , Retinoid X Receptors/biosynthesis , Animals , Corticosterone/blood , Disease Susceptibility , Insulin/blood , Liver Neoplasms/etiology , Liver X Receptors , Male , Mice , Mice, Inbred Strains , Species SpecificityABSTRACT
The carcinogenic effects of estragole in mice of the earlier unexplored strain ICR has been studied. It has been shown that there is a distinct correlation between the extent of inhibition of glucocorticoid-mediated induction of tyrosine aminotransferase and trypthophan oxygenase after acute administration of estragole and the frequency of liver tumors after estragole exposure. Estragole inhibits the induction of these enzymes only in female mice, but not in male mice and rats. DNA-binding activities of liver-enriched transcription factors were investigated on carcinogen-susceptible and -resistant animals. Estragole decreases the HNF4 (hepatic nuclear factor 4) and FOXA DNA-binding activities only in susceptible female mice, but not in nonsusceptible male mice and rats and does not influence the C/EBP and HNF1 activities. Pentachlorophenol, which prevents the hepatocarcinogenic effect of estragole, abolishes its inhibitory effect on tyrosine aminotransferase and trypthophan oxygenase glucocorticoid induction and restores the FOXA and HNF4 DNA-binding activities. The parallelism between the hepatocarcinogenic effects of estragole and the inhibition of FOXA and HNF4 DNA-binding activities serves as an additional argument for the involvement of these factors in the mechanisms of tumor suppression in the liver.
Subject(s)
Anisoles/toxicity , Carcinogens/toxicity , Glucocorticoids/pharmacology , Hepatocyte Nuclear Factor 4/metabolism , Liver Neoplasms/metabolism , Liver/enzymology , Neoplasm Proteins/metabolism , Allylbenzene Derivatives , Animals , Enzyme Induction/drug effects , Enzyme Inhibitors/pharmacology , Female , Liver Neoplasms/chemically induced , Male , Mice , Mice, Inbred ICR , Organ Specificity/drug effects , Pentachlorophenol/pharmacology , Rats , Rats, Wistar , Sex CharacteristicsABSTRACT
The content of peroxisome proliferation activating proteins PPAR-alpha and PPAR-gamma, liver X receptors (LXR), and retinoid X receptors (RXR) and activity of PPAR-alpha, PPAR-gamma, and PPAR-delta binding to DNA response elements in C57Bl/6 mouse macrophages were studied during different phases of aseptic inflammation, induced by intraperitoneal injection of 50 mg/kg zymosan A. The DNA-binding activities of PPAR-alpha and PPAR-gamma and the levels of PPAR-alpha, PPAR-gamma, LXR, and RXR in peritoneal macrophages dropped on days 1 and 3 after zymosan injection. On days 7 and 14 the DNA-binding activity of PPAR-gamma and content of PPAR-gamma and LXR-beta protein increased in comparison with the control, while the DNA-binding activity and content of PPAR-alpha in the cells remained low. Recovery of RXR protein content in macrophages was observed only on day 14 after zymosan injection.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Inflammation/metabolism , Macrophages/metabolism , Peroxisome Proliferator-Activated Receptors/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Retinoid X Receptors/metabolism , Animals , Immunoblotting , Inflammation/chemically induced , Liver X Receptors , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Orphan Nuclear Receptors , Protein Binding/drug effects , Tumor Necrosis Factor-alpha/metabolism , Zymosan/pharmacologyABSTRACT
It is known that the carcinogenic effect of estragole, a component of essential oils of many spicy plants, is characterized by species, tissue, and sex specificity. It causes mainly liver tumors in female mice but is not carcinogenic for male mice and for rats. In this work, the estragole hepatocarcinogenicity was shown for female mice of previously not studied ICR line. The strict correlation between estragole hepatocarcinogenicity and its ability to decrease the level of glucocorticoid induction of liver-specific enzymes tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) was found. Inhibition of TAT and TO inducibility by estragole takes place only in female mice but not in male mice and in rats. Studying the estragole effect on DNA-binding activity of transcription factors, present mainly in liver and regulating expression of genes encoding liver-specific proteins, has shown that estragole decreases FOXA and HNF4 activities but not activities of C/EBP and HNF1, and this happens only in female mice, for which this substance is hepatocarcinogen, but not in male mice and in rats. Pentachlorophenol, preventing hepatocarcinogenic effect of estragole, abolishes inhibitory influence of the latter on the TAT and TO glucocorticoid induction and restores DNA-binding activity of FOXA and HNF4. Thus, a correlation was revealed between the estragole hepatocarcinogenic effect and decrease in DNA-binding activity of transcription factors FOXA and HNF4, which might be indicative of the role of these factors in tumor suppression mechanisms in liver.
Subject(s)
Anisoles/pharmacology , Carcinogens/pharmacology , Forkhead Transcription Factors/metabolism , Glucocorticoids/metabolism , Hepatocyte Nuclear Factor 4/metabolism , Liver/drug effects , Liver/enzymology , Allylbenzene Derivatives , Animals , Female , Forkhead Transcription Factors/genetics , Hepatocyte Nuclear Factor 4/genetics , Liver/metabolism , Male , Mice , Mice, Inbred ICR , Rats , Rats, Wistar , Sex Characteristics , Species Specificity , Tryptophan Oxygenase/metabolism , Tyrosine Transaminase/metabolismABSTRACT
Changes in electrical charge and clearance rate of LDL after the formation of their complexes with bacterial LPS were studied in experiments on Wistar rats. It was found that binding of S. minnesota R595 LPS with (125)I-LDL sharply accelerated clearance of the greater part of LDL complexes, but on the other hand induced the appearance of an LDL-LPS subfraction with slower elimination rate compared to free LDL. Electrophoresis showed that after binding of LPS, LDL acquired a negative charge. These data suggest that the formation of LDL-LPS complexes is accompanied by modification of LDL due to which they acquire atherogenic properties.
Subject(s)
Endotoxins/metabolism , Lipoproteins, LDL/pharmacokinetics , Lipoproteins/metabolism , Animals , Electrophysiology , Endotoxins/blood , Half-Life , Iodine Radioisotopes/pharmacokinetics , Lipopolysaccharides/blood , Lipopolysaccharides/metabolism , Lipoproteins/blood , Lipoproteins, LDL/blood , Lipoproteins, LDL/chemistry , Male , Metabolic Clearance Rate , Multiprotein Complexes/blood , Multiprotein Complexes/metabolism , Protein Binding , Protein Processing, Post-Translational/physiology , Rats , Rats, WistarABSTRACT
The effects of peroxisome proliferator activated receptors alpha and gamma (PPAR-alpha and PPAR-gamma) and retinoid X receptor (RXR) agonists upon synthesis and accumulation of lipids in murine C57Bl macrophages during inflammation induced by injection of zymosan and Escherichia coli lipopolysaccharide (LPS) have been studied. It is significant that intraperitoneal injection of zymosan (50 mg/kg) or LPS (0.1 mg/kg) in mice led to a dramatic increase of [14C]oleate incorporation into cholesteryl esters and triglycerides and [14C]acetate incorporation into cholesterol and fatty acids in peritoneal macrophages. Lipid synthesis reached its maximum rate 18-24 h after injection and was decreased 5-7 days later to control level after LPS injection or was still heightened after zymosan injection. In macrophages obtained in acute phase of inflammation (24 h), degradation of 125I-labeled native low density lipoprotein (NLDL) was 4-fold increased and degradation of 125I-labeled acetylated LDL (AcLDL) was 2-3-fold decreased. Addition of NLDL (50 microg/ml) or AcLDL (25 microg/ml) into the incubation medium of activated macrophages induced 9-14- and 1.25-fold increase of cholesteryl ester synthesis, respectively, compared with control. Addition of NLDL and AcLDL into the incubation medium completely inhibited cholesterol synthesis in control macrophages but had only slightly effect on cholesterol synthesis in activated macrophages. Injection of RXR, PPAR-alpha, or PPAR-gamma agonists--9-cis-retinoic acid (5 mg/kg), bezafibrate (10 mg/kg), or rosiglitazone (10 mg/kg), respectively--30 min before zymosan or LPS injection led to significant decrease of lipid synthesis. Ten hour preincubation of activated in vivo macrophages with the abovementioned agonists (5 microM) decreased cholesteryl ester synthesis induced by NLDL and AcLDL addition into the cell cultivation medium. The data suggest that RXR, PPAR-alpha, or PPAR-gamma agonists inhibited lipid synthesis and induction of cholesteryl ester synthesis in inflammatory macrophages caused by capture of native or modified LDL.
Subject(s)
Inflammation/metabolism , Lipids/biosynthesis , Macrophages, Peritoneal/metabolism , Peroxisome Proliferator-Activated Receptors/agonists , Retinoid X Receptors/agonists , Acetic Acid/metabolism , Animals , Cholesterol/biosynthesis , Cholesterol/chemistry , Cholesterol Esters/biosynthesis , Cholesterol Esters/chemistry , Fatty Acids/biosynthesis , Inflammation/chemically induced , Lipopolysaccharides/administration & dosage , Lipoproteins, LDL/metabolism , Macrophages, Peritoneal/drug effects , Male , Mice , Mice, Inbred C57BL , Oleic Acid/metabolism , PPAR alpha/agonists , PPAR gamma/agonists , Triglycerides/biosynthesis , Triglycerides/chemistry , Zymosan/administration & dosageABSTRACT
A single intraperitoneal injection of Estragole (300 mg/kg) to female ICR mice 19 hours prior to Dexamethasone induction decreased induced activities of tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) nearly to 50% of the control values. In these mice, activities of the marker enzymes of liver damage: alanine aminotransferase (ALAT) and aspartate aminotransferase (AAT) increased in the blood 1.7-2.3-fold as compared with the untreated controls. By contrast, carbon tetrachloride (100 mg/kg) increased the blood AIAT and AsAT activities 135- and 30-fold as compared with the control, but inhibited the TAT and TO induction much less than Estragole did. Estragole seems to inhibit the glucocorticoid induction of these hepatic enzymes not via the unspecific toxic damage of the liver.
Subject(s)
Anisoles/toxicity , Carcinogens/toxicity , Liver/drug effects , Tryptophan Oxygenase/antagonists & inhibitors , Tyrosine Transaminase/antagonists & inhibitors , Alanine Transaminase/metabolism , Allylbenzene Derivatives , Animals , Anisoles/administration & dosage , Aspartate Aminotransferases/metabolism , Carcinogens/administration & dosage , Dexamethasone/pharmacology , Enzyme Activation , Female , Injections, Intraperitoneal , Liver/enzymology , Mice , Mice, Inbred ICR , Rats , Rats, Sprague-Dawley , Species Specificity , Tryptophan Oxygenase/biosynthesis , Tyrosine Transaminase/biosynthesisABSTRACT
It is well known that artificial increase in expression of growth factors and their receptors can lead to tumorigenic transformation of cells. Additionally, multiple data on the increased expression of growth factors in many human and animal tumorigenic cells have been published. Nevertheless description of the functional role of endogenous growth factors in maintenance of tumorigenic phenotype remains obscure. Previously, we described a new model for studying neoplastic transformation and dormant metastasis. This model consists of cognate tumorigenic and nontumorigenic cell clones, the latter being obtained as a result of spontaneous reversion of tumorigenic ones. All revertant clones demonstrate the three well known features of normal cells: monolayer growth on a plastic, incapability to grow in soft agar with regular culture media with 10% FCS, and incapability to form tumors in syngeneic animals. Using RT-PCR, we measured expression ratios of main growth factors, commonly believed to be associated with malignization, in tumorigenic and revertant clones of our model. For some of these clones, we also measured an activity of growth factors in conditioned media. The data obtained argue that the levels of growth factor expression, measured by both the methods, are distributed between tumorigenic and revertant clones in a sporadic manner and do not correlate with cell tumorigenicity. Thus, our experimental observations enable us to consider the variability of growth factor expression as insignificant events in the reversion of tumorigenic cells to a nontumorigenic phenotype.
Subject(s)
Clone Cells/metabolism , Growth Substances/metabolism , Animals , Base Sequence , Culture Media, Conditioned , DNA Primers , Humans , Mice , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
Unlike other cytochrome P450-dependent oxygenase inhibitors, ketoconazole has been shown to suppress the murine macrophage-mediated oxidative modification of human low-density lipoproteins (LDL) in a dose-dependent manner. The benzo[alpha]pyrene-induced microsomal monooxygenase activity was accomplished by a 1,5-fold increase in LDL oxidation by macrophages, ketokonazole (20 mu), methoxalene (20 mu), and alpha-naphthaflavone (50 mu). Ketoconazole was also effective in inhibiting macrophageal NADPH oxidase and LDL autooxidation induced by Fe2+ rather than Cu+, which is likely to be associated with its ability to act as a chelator of free and heme-bound iron ions.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Lipoproteins, LDL/metabolism , Macrophages, Peritoneal/metabolism , Animals , Benzo(a)pyrene/pharmacology , Benzoflavones/pharmacology , Copper/pharmacology , Ferrous Compounds/pharmacology , Humans , Iron Chelating Agents/pharmacology , Ketoconazole/pharmacology , Luminescent Measurements , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/enzymology , Methoxsalen/pharmacology , Mice , NADPH Oxidases/antagonists & inhibitors , Oxidation-ReductionABSTRACT
Known cytochrome P450-dependent oxygenase inhibitor ketoconazole (5-50 microM) blocked the murine macrophage-mediated modification of human low density lipoprotein (LDL) as measured by production of thiobarbituric acid-reactive substance, stimulation of [125I]LDL degradation in a fresh set of macrophages and LDL electrophoretic mobility, in a dose-dependent manner with complete inhibition at 30-40 microM. When resident macrophages were incubated with LDL in the presence of metyrapone, methoxsalen and alpha-naphthaflavone at concentrations that have been shown to inhibit the cytochrome P450-dependent oxygenases, there was no change in LDL modification. Induction of benzo[alpha]pyrene hydroxylase activity in macrophages by 24 h incubation with benzo[alpha]pyrene was accompanied by a 1.5-fold increase of LDL modification which has been leveled down by ketoconazole as well as methoxsalen and alpha-naphthaflavone. Furthermore, ketoconazole effectively diminished cell-free LDL oxidation induced by iron, but not copper ions, and reduced the spontaneous and zymosan-stimulated lucigenin-amplified chemiluminescence of macrophages. The data allow us to suggest that ketoconazole inhibits LDL oxidation by acting as an iron chelator and/or inhibitor of prooxidant forms of iron-containing enzymes.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Ketoconazole/pharmacology , Lipoproteins, LDL/antagonists & inhibitors , Macrophages, Peritoneal/drug effects , Animals , Benzoflavones/pharmacology , Benzopyrene Hydroxylase/pharmacology , Cells, Cultured , Cytochrome P-450 Enzyme System/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Humans , Lipoproteins, LDL/metabolism , Luminescent Measurements , Macrophages, Peritoneal/metabolism , Methoxsalen/pharmacology , Metyrapone/pharmacology , Mice , Mice, Inbred Strains , Oxidation-Reduction/drug effectsABSTRACT
The effect of polyphenols (PP) from Alchimilia vulgaris L. and Rosa majalis Herrm. on oxidative modification of low density lipoproteins (LDL) was studied in the cultured peritoneal macrophages and in unicellular medium in the presence of Cu2+. The experiments on cultured macrophages indicated that PP (10-50 micrograms/ml) reduced the lipid peroxidation products, measured as thiobarbituric acid reactive substances, by as much as 40-77% and decreased the macrophage degradation of 125I-LDL by 60-87%. In contract, the addition of alpha-tocopherol (50-100 microM) inhibited by only 40-60% under the same conditions. Oxidation of LDL by macrophages was effectively dose-dependently inhibited by PP (5-500 micrograms/ml), as was Cu(2+)-catalyzed oxidation of LDL. Our data indicate that PP of these plants protect LDL against oxidation and may be used for the prevention and regression of atherosclerosis.