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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.

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.

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.

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.

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.

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.

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): 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.

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.

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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Journal Article 2008; 29(5): 728-732 PubMed PMID: 18987592 Citation  Keywords:  Acetyl Coenzyme A:metabolism, Arylamine N-Acetyltransferase:metabolism, Arylsulfotransferase:metabolism, Autoradiography, Catalysis, DNA:chemistry, DNA Adducts:chemistry, Deoxyguanosine:chemistry, Ellipticines:chemistry, Hydrogen-Ion Concentration, Indica.   

OBJECTIVES: Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of its action. Recently, we have found that 13-hydroxyellipticine, formed from ellipticine as the predominant metabolite in human livers, is bound to deoxyguanosine in DNA, generating the major DNA adduct in vivo and in vitro. The development of the methods suitable for the preparation of this adduct in the amounts sufficient for identification of its structure and those for its isolation and partial characterization is the aim of this study.

METHODS: High performance liquid chromatography (HPLC) was employed for separation of 13-hydroxyellipticine-mediated deoxyguanosine adduct. The 32P-postlabeling technique was utilized to detect this adduct in DNA.

RESULTS: The formation of the 13-hydroxyellipticine-derived deoxyguanosine adduct in DNA in vitro was increased under the alkaline pH of the incubations and by the formation of the sulfate and acetate conjugates of 13-hydroxyellipticine generated by reactions with 3'-phosphoadenosine-5'-phosphosulfate (PAPS) or acetyl-coenzyme A (acetyl-CoA) catalyzed by human sulfotransferases (SULTs) 1A1 and 1A2 and N,O-acetyltransferases (NATs) 1 and 2. The HPLC method suitable for separation the 13-hydroxyellipticine-derived deoxyguanosine adduct from other reactants, deoxyguanosine and 13-hydroxyellipticine, was developed. The structure of this adduct is proposed to correspond to the product formed from ellipticine-13-ylium with the exocyclic 2-NH2 group of guanine in DNA.

CONCLUSIONS: The data are the first report on HPLC isolation of the deoxyguanosine adduct formed by 13-hydroxyellipticine in DNA and its partial characterization....

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Journal Article 2008; 29(5): 675-679 PubMed PMID: 18987591 Citation  Keywords:  Adolescent, Age Factors, Animals, Child, Czech Republic:epidemiology, Dental Amalgam, Dental Restoration, Permanent, Diet, Female, Fishes:metabolism, Hair:chemistry, Humans, Male, Meat, Mercury:analysis, Poland:epidemiology, Sex Factors,.   

OBJECTIVES: Mercury and most of its compounds are extremely toxic and should be handled with care. It can be inhaled and absorbed through the skin and mucous membranes. The most toxic forms of mercury are its organic compounds such as dimethylmercury and methylmercury. Fish have a natural tendency to accumulate mercury. Methylmercury is produced by microbial methylation of inorganic mercury in water sediment then it infiltrates the food chain and it consequently accumulates in fish. Fish are the main source of methylmercury in human food. Mercury is transferred into a hair; and this can be than used to monitor the long-term exposure to mercury. The content of mercury in hair depends on the frequency of fish consumption. The aim of our study was to compare mercury content in the hair of children that had various amounts of fish consumption (increased or reduced).

DESIGN: Total mercury content in hair was determined by direct method of cold vapors using an AMA 245 analyzer. A total of 174 hair samples from the children (9-17 years old) were analyzed. In this study, the following localities were compared: Neratovice (n=42), Jeseníky (n=44), Prague (n=59) in Czech Republic and Olsztyn in Poland (n=29). Every sample was accompanied with questionnaire about age, gender, regions, amalgam fillings and fish consumption.

RESULTS: We did not find a correlation between the content of mercury in hair with age, gender or amalgam fillings. We did find a correlation between fish consumption and the amount of mercury found in the hair samples.

CONCLUSION: The amount of mercury in hair increases with more frequent consumption of freshwater and marine fish....

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Comparative Study 2008; 29(5): 755-758 PubMed PMID: 18987590 Citation  Keywords:  Acetylcholinesterase:chemistry, Algorithms, Alzheimer Disease:drug therapy, Biosensing Techniques, Cholinesterase Inhibitors:analysis, Electrochemistry, Enzymes, Immobilized:chemistry, Humans, Kinetics, Nootropic Agents:analysis, Tacrine:analogs & derivat.   

: Two drugs were tested using electrochemical biosensor with immobilized acetylcholinesterase (AChE). The first was commercialized drug tacrine (known also as Cognex) used for treatment of cognitive manifestation of Alzheimer\'s disease (AD). The second one was its 7-methoxy derivate (7-MEOTA) that has not been marketed. We determined the IC50 (6.67+/-0.92)x10-7 M for tacrine and (1.66+/-1.43)x10-9 M for 7-MEOTA. In this in vitro study, 7-MEOTA acts as stronger inhibitor of AChE and in this way could be more favorable for treatment of cognitive manifestation of AD. Our study shows that biosensor technology could be used as a quick and cheap tool for testing of promising AChE inhibitors (AD drug candidates)....

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Journal Article 2008; 29(5): 706-711 PubMed PMID: 18987589 Citation  Keywords:  Animals, Antioxidants:therapeutic use, Arthritis, Experimental:blood, Chromatography, High Pressure Liquid, Enzyme-Linked Immunosorbent Assay, Indoles:therapeutic use, Lipid Peroxidation:drug effects, Male, Mass Spectrometry, Muscle, Skeletal:drug effects.   

OBJECTIVES: To study possible oxidation of proteins and lipids in plasma and sarcoplasmic reticulum (SR) from skeletal muscles and to assess the effects of pyridoindole antioxidants in rats with adjuvant arthritis (AA) and to analyze modulation of Ca-ATPase activity from SR (SERCA).

METHODS: SR was isolated by ultracentrifugation, protein carbonyls in plasma and SR were determined by ELISA. Lipid peroxidation was analyzed by TBARS determination and by mass spectrometry. ATPase activity of SERCA was measured by NADH-coupled enzyme assay. Tryptophan fluorescence was used to analyze conformational alterations.

RESULTS: Increase of protein carbonyls and lipid peroxidation was observed in plasma of rats with adjuvant arthritis. Pyridoindole antioxidant stobadine and its methylated derivative SMe1 decreased protein carbonyl formation in plasma, effect of stobadine was significant. Lipid peroxidation of plasma was without any effect of pyridoindole derivatives. Neither protein oxidation nor lipid peroxidation was identified in SR from AA rats. SERCA activity from AA rats increased significantly, stobadine and SMe1 diminished enzyme activity. Ratio of tryptophan fluorescence intensity in SR of AA rats increased and was not influenced by antioxidants.

CONCLUSION: Plasma proteins and lipids were oxidatively injured in rats with AA; antioxidants exerted protection only with respect to proteins. In SR, SERCA activity was altered, apparently induced by its conformational changes, as supported by study of tryptophan fluorescence. Stobadine and SMe1 induced a decrease of SERCA activity, elevated in AA rats, but they did not affect conformational changes associated with tryptophan fluorescence....

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Journal Article 2008; 29(5): 649-652 PubMed PMID: 18987588 Citation  Keywords:  Animals, Antineoplastic Agents, Phytogenic:pharmacology, Antioxidants:pharmacology, Apoptosis:drug effects, Biphenyl Compounds, Chelidonium:chemistry, Dose-Response Relationship, Drug, Free Radical Scavengers, HL-60 Cells, Humans, Leukemia:drug therapy, L.   

OBJECTIVES: The purpose of this study was to assess whether a methanol extract isolated from the greater celandine Chelidonium majus L. (CME) had antioxidant effect and was able to inhibit proliferation and to induce apoptosis in leukemia cells in vitro.

METHODS: The potential antioxidant activity of CME was proved by the 1,1-diphenyl- 2-picrylhydrazyl (DPPH) radical scavenging assay. The cytotoxicity of CME was measured by the cell growth inhibition assay using murine leukemia L1210 cell line and human promyelocytic HL-60 leukemia cells. Apoptosis-inducing effect was determined by fluorescence microscopy (chromatin condensation and nuclear DNA fragmentation).

RESULTS: In the DPPH assay CME acted as a scavenger of DPPH free radical. The results on antiproliferative properties assessment clearly demonstrated that CME had a cytotoxic effect towards both leukemia cell lines in a dose-dependent manner. In addition, the human promyelocytic HL-60 cells were more sensitive to CME treatment than the L1210 cells.

CONCLUSIONS: We concluded that the extract of C. majus L. had a strong antioxidant potential and exerted the antiproliferative activity via apoptosis on leukemia cells. CME due to the presence of the isoquinoline alkaloids and the flavonoid components may play an important role in both cancer chemoprevention through its antioxidant activity and modern cancer chemotherapy as cytotoxic and apoptosis-inducing agent....

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