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Journal Article 2008; 29(5): 644-648 PubMed PMID: 18987600 Citation  Keywords:  Animals, Antioxidants, Apoptosis:drug effects, Cell Count, Cell Death, Cell Separation, Chitin:analogs & derivatives, Comet Assay, DNA:drug effects, DNA Damage:drug effects, Female, Glucans:pharmacology, Hepatocytes:drug effects, Hydrogen Peroxide:toxicit.   

OBJECTIVES: Utilizing primary rat hepatocytes we investigated the potential antimutagenic and anti-cytotoxic effects of carboxymethyl chitin-glucan (CM-CG) with respect to oxidative stress induced by the model free-radical-generating compounds hydrogen peroxide (H2O2) or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). Different kinds of CM-CG action were studied by two different treatment protocols: a. pre-incubation of freshly isolated hepatocytes with the potential anti-mutagen followed by exposure to the oxidant or b. simultaneous treatment of hepatocytes with the potential anti-mutagen and the oxidant.r />r /> METHODS: As a measure of genotoxicity, the percentages of DNA in tails of comets by single cell gel electrophoresis were evaluated. The cytotoxicological endpoints analysed were the cell density (number of cells/cm2), and the percentages of apoptotic and necrotic cells.r />r /> RESULTS: H2O2 and DMNQ, causing DNA single-strand breaks via the formation of *OH radicals, have been demostrated to induce both genotoxic and cytotoxic effects in primary rat hepatocytes resulting in increased percentages of DNA in tails of comets, and increased frequencies of apoptotic and necrotic cells accompanied by a decreased cell density. Further investigations were therefore focussed on possible modifications of these parameters by CM-CG. The results obtained clearly demonstrate that CM-CG (applied before and during treatment) protects primary rat hepatocytes against the genotoxic and cytotoxic effects of oxidative stress (H2O2 or DMNQ), whereas CM-CG itself has no effect on the endpoints of genotoxicity and cytotoxicity studied.r />r /> CONCLUSION: Our results indicate that carboxymethyl chitin-glucan represents a natural fungal polysaccharide that can inhibit the genotoxicity and cytotoxicity of experimentally induced oxidative stress in primary rat hepatocytes....

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Journal Article 2008; 29(5): 691-696 PubMed PMID: 18987599 Citation  Keywords:  Animals, Anti-Inflammatory Agents, Non-Steroidal, Antioxidants:metabolism, Arthritis, Experimental:pathology, Candida:chemistry, Foot:pathology, Inflammation:pathology, Male, Mannans:pharmacology, Mycobacterium:immunology, Oxidative Stress:drug effects, P.   

OBJECTIVES: The aim of this study was to evaluate the effects of a biological response modifier, glucomannan (GM), isolated from Candida utilis, on the progress of adjuvant arthritis in Lewis rats.r />r /> METHODS: Adjuvant arthrithis was induced in Lewis rats by a single intradermal injection of Mycobacterium butyricum. GM was administered in two different doses of 5 and 7.5 mg/kg b.w. The treatment involved daily oral or intraperitoneal administration of the substance from day 0, i.e. the day of immunization to the end of the experiment - day 28. Cyclosporin A was used as a therapeutic standard in daily oral dose of 2.5 mg/kg b.w. The following parameters were monitored: hind paw volume, total antioxidant status, protein carbonyl groups, activity of N-acetyl-beta-D-glucosaminidase in plasma, lysozyme and peroxidase activity of peritoneal macrophages and activity of gamma-glutamyltransferase in homogenates of spleen, hind paw muscle and hind paw joint.r />r /> RESULTS: Beneficial action of GM was revealed mainly in hind paw volume decrease. Further decrease of the activity of the enzyme gamma glutamyltransferase (GGT) in the spleen, hind paw joint and muscle tissue homogenates, decrease of the plasmatic activity of N-acetyl-beta-D-glucosaminidase (NAGA), and finally suppression of lysozyme and peroxidase activity assessed in peritoneal macrophages were observed in arthritic animals treated with GM. All these findings speak in favor of the anti-inflammatory activity of glucomannan. Moreover, a significant improvement of the arthritis induced suppression of total antioxidant status and decrease of the level of the arthritis-associated protein carbonyls in plasma were detected.r />r /> CONCLUSIONS: The important characteristics of GM isolated from Candida utilis, such as good water solubility and relatively small molecular weight, along with the observed in vivo anti-inflammatory and antioxidant effects, appear to be promising features for its prospective use as a natural agent in prevention and supplementary therapy of rheumatoid arthritis....

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Journal Article 2008; 29(5): 733-737 PubMed PMID: 18987598 Citation  Keywords:  Animals, Aristolochic Acids:pharmacokinetics, Carcinogens:pharmacokinetics, Chromatography, High Pressure Liquid, Cytochrome P-450 CYP1A1:isolation & purification, Cytochrome P-450 CYP1A2:isolation & purification, Humans, Inactivation, Metabolic, Mice, Mi.   

OBJECTIVES: A carcinogenic and nephrotoxic plant alkaloid, aristolochic acid (AA), causes the development of aristolochic acid nephropathy, which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA may also cause a similar type of kidney fibrosis with malignant transformation of the urothelium, the Balkan endemic nephropathy. The aim of the study was to resolve which cytochromes P450 (CYP) detoxicate the major component of AA, aristolochic acid I (AAI), to its O-demethylated metabolite, aristolochic acid Ia (AAIa).r />r /> METHODS: High performance liquid chromatography (HPLC) was employed for separation and characterization of AAI metabolites generated by CYPs.r />r /> RESULTS: Human, rat and mouse hepatic CYPs oxidize AAI into its detoxication metabolite AAIa. Most of the detoxication of AAI in human hepatic microsomes is mediated by CYP1A2 and 1A1, while other CYPs play a minor role.r />r /> CONCLUSIONS: The data are the first report on identification of human CYP enzymes detoxicating AAI....

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Journal Article 2008; 29(5): 685-690 PubMed PMID: 18987597 Citation  Keywords:  Cartilage:cytology, Cell Differentiation:drug effects, Chondrocytes:drug effects, Collagen Type II:biosynthesis, Collagen Type XI:biosynthesis, Extracellular Matrix:drug effects, Gene Expression:drug effects, Glycosaminoglycans:metabolism, Humans, Hyaluro.   

OBJECTIVES: Hyaluronan (HA) is an abundant component of chondrogenic tissue hence it is often used as a fundamental constituent in cartilage tissue substitutes. However, effects of different molecular weight HA on chondrogenic differentiation are not clear. The aim was to evaluate modulation of mesenchymal stem cell (MSC) early chondrogenesis by HA of molecular weights 100, 600 and 1 500 kDa.r />r /> METHODS: HA was applied on MSCs cultured in a pellet system for one, two and three weeks. Chondrogenesis was evaluated by determinations of gene expression of transcription factor Sox-9 and extracellular matrix proteins collagen type II and XI, aggrecan, and COMP by Real-Time PCR and completed with histological analysis.r />r /> RESULTS: Upon chondrogenic induction, the respective pellets revealed active transcription of the chondrogenic genes together with proceeding accumulation of glycosaminoglycan (GAG) rich extracellular matrix. Sox-9 was also expressed in non-chondrogenic MSC controls. HA treated pellets were not significantly influenced on day 7 of culture. However, on day 14, lowered expression in some of the extracellular matrix proteins appeared together with a moderately smaller amount of GAG content in pellet sections. Nevertheless, the analysis on day 21 has demonstrated that HA did not affect the outcome of the differentiation by the end of the culture. Any difference regarding the molecular weight of the HA was not found.r />r /> CONCLUSIONS: It could be speculated that HA induced a time shift in the phase of the dominant matrix protein onset which was in full compensated by the end of the evaluated time period. Thus, data suggest that HA of any tested molecular weight does not significantly modulate chondrogenesis of MSCs in pellet system....

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Journal Article 2008; 29(5): 717-721 PubMed PMID: 18987596 Citation  Keywords:  Allyl Compounds:pharmacology, Animals, Anticarcinogenic Agents:pharmacology, Blotting, Western, Curcumin:pharmacology, Cytochrome P-450 CYP1A1:biosynthesis, Cytochrome P-450 CYP2B1:biosynthesis, Cytochrome P-450 Enzyme System:biosynthesis, Flavonoids:phar.   

OBJECTIVES: Since flavonoids and other natural compounds exert beneficial effects on human health, their consumption rapidly increases. However, they can modulate the activity of xenobiotic-metabolizing enzymes involved in activation and detoxification of food and environmental carcinogens. Thus, their potential negative effects should be examined.r />r /> METHODS: The induction effects of selected chemopreventive compounds, administered per orally by gastric gavages to rats, on cytochrome P450 (CYP) 1A and 2B were determined in liver and small intestine using Western blotting analysis and specific metabolic activity assays.r />r /> RESULTS: Comparing CYPs expression along small intestine, the highest induction was observed in the proximal part near pylorus with rapid decrease towards the distal part. In response to chemopreventive compounds, the marked induction of CYP1A and CYP2B in liver was observed after diallyl sulphide and flavone treatment. In small intestine, beta-naphthoflavone, diallyl sulphide and curcumin induced CYP1A1 and CYP2B1. In both tissues, resveratrol did not significantly affect CYPs expression. The results of Western blotting detection of CYPs correlate well with their specific enzymatic activities.r />r /> CONCLUSIONS: Presented data indicate ambiguous impact of chemopreventive compounds on cytochromes P450, main xenobiotic-metabolizing enzymes. Thus, the question of safety and unlimited consumption of these compounds arises....

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Journal Article 2008; 29(5): 790-793 PubMed PMID: 18987595 Citation  Keywords:  Adrenergic beta-Antagonists:pharmacology, Androstadienes:pharmacology, Calcimycin:pharmacology, Carbazoles:pharmacology, Humans, In Vitro Techniques, Male, N-Formylmethionine Leucyl-Phenylalanine:pharmacology, Neutrophils:drug effects, Oxidants:metabolism.   

OBJECTIVES: To compare three stimuli which activate human neutrophils with different signal transduction mechanisms, in order to better localize the effect of the beta-adrenoceptor antagonist carvedilol (CARV) on superoxide generation (O2*-) and myeloperoxidase release (MPO). The effect of CARV [0.1-100 micromol/l] on O2*- generation and MPO release from isolated human neutrophils was studied after specific receptor activator N-formyl-methionyl-leucyl-phenylalanine (fMLP) and nonreceptor phorbol-12-myristate-13-acetate (PMA) and calcium ionophor (A23187) stimuli.r />r /> METHODS: O2*- generation was measured as superoxide dismutase inhibitable reduction of cytochrome c and MPO release as the oxidation of o-dianisidine in the presence of hydrogen peroxide in a spectrophotometer Hewlet Packard 8452 A at respective 550 and 463 nm.r />r /> RESULTS: CARV had no effect on O2*- generation and MPO release in nonstimulated cells. In the concentration 10 and 100 micromol/l, it significantly decreased fMLP and PMA stimulated O2*- generation and MPO release. Incubation of neutrophils with CARV [100 micromol/l] caused significant inhibition of O2*- generation and MPO release induced by A23187. Wortmannin, a specific inhibitor of 1-phosphatidylinositol-3-kinase, inhibited significantly only fMLP stimulated O2*- generation. CARV [100 micromol/l] with wortmannin [50 nmol/l] further decreased O2*- generation after the same stimulus.r />r /> CONCLUSION: CARV decreased O2*- generation and MPO release from isolated human neutrophils both by membrane-operating stimulus - fMLP and membrane bypassing activators - PMA and A 23187. This fact, together with effect the of wortmannin, indicates that the inhibition may be attributed to the non-specific action of CARV and its interference with phospholipase D signaling pathway, which plays only a minor role in proteinkinase C stimulated O2*- generation....

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Comparative Study 2008; 29(5): 738-743 PubMed PMID: 18987594 Citation  Keywords:  Animals, Dogs, Glucuronides:metabolism, Glucuronosyltransferase:metabolism, Haplorhini, Humans, Kinetics, Microsomes, Liver:enzymology, Protective Agents:metabolism, Rats, Silymarin:metabolism, Species Specificity, Swine,.   

OBJECTIVES: The study of interspecies differences in glucuronidation processes in the man, monkey, pig, dog and rat using liver microsomal fraction. The study is focused on determination of the enzyme activity of UGT1A6 (having also a toxicological importance) in microsomes of different species.r />r /> METHODS: For determination of glucuronides formed, an HPLC method with UV detection and LC-MS characterization was used. p-Nitrophenol and 4-methylumbelliferon and silybin were chosen as model substrates.r />r /> RESULTS: The data presented in this paper show an overall similarity in kinetic parameters of the UGT1A6 with p-nitrophenol and 4-methylumbelliferon for man, pig and monkey. The pattern of silybin glucuronides formed in monkey and dog samples are relatively close to this of the man.r />r /> CONCLUSIONS: For studies of glucuronidation of xenobiotics where the role UGT1A6 is expected, the use of pig and monkey microsomes should be considered. As an optimal model for study of silybin glucuronidation, both the rhesus monkey and dog (Beagle) seem to be the best models. To elucidate the role of the UGT forms involved in metabolism of silybin, the experiments with recombinant UGT enzymes are needed....

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Journal Article 2008; 29(5): 744-748 PubMed PMID: 18987593 Citation  Keywords:  Amanita:chemistry, Amanitins:urine, Chromatography, High Pressure Liquid, Humans, Ibotenic Acid:urine, Indicators and Reagents, Muscarine:urine, Muscimol:urine, Nucleic Acid Synthesis Inhibitors:urine, Solid Phase Extraction, Solvents, Spectrometry, Mass,.   

OBJECTIVES: Ibotenic acid, muscimol and muscarine were recognized as responsible for psychotropic effects of A. muscaria and A. pantherina. Demand for their specific and sensitive identification and quantitation in biological material lead to effort to develop reliable analytical method, but satisfactory solution is still lacking. Presented article describes liquid chromatography-mass spectrometry method suitable for isolation and identification of principal toxins of A. muscaria and A. pantherina in urine.r />r /> METHODS AND RESULTS: Dedicated liquid chromatography-mass spectrometry method is reported. Technique consists of an extraction of analytes on Strata X-CW and Discovery SCX SPE cartridges and separation is achieved using a Gemini C18 column (150 mm x 2.0 mm, 5 micron) and 8 mM heptafluorobutyric acid as mobile phase. Detection at m/z 159 for ibotenic acid, m/z 115 for muscimol and m/z 174 for muscarine was used. Retention times and LODs are 2.6 min and 50 ng.ml-1 for ibotenic acid, 4.6 min and 40 ng.ml-1 for muscimol and 14.2 min and 3 ng.ml-1 for muscarine.r />r /> CONCLUSION: A sensitive and specific liquid chromatography-mass spectrometry assay was developed for analysis of principal toxins of A. muscaria and A. pantherina in urine. Method was found to be sufficiently sensitive and specific for analysis of urine of intoxicated patients....

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