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Journal Article 2009; 30(Suppl 1): 60-66 PubMed PMID: 20027146 Citation  Keywords:  Antineoplastic Agents:administration & dosage, Autoradiography, Blotting, Western, Cell Line, Tumor, Cell Proliferation:drug effects, Cell Survival:drug effects, Chromatography, High Pressure Liquid, Cytochrome P-450 Enzyme System:metabolism, DNA Adducts:.   

OBJECTIVES: Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action with promising brain tumor specificity. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP) - and/or peroxidase-mediated activation to species forming covalent DNA adducts. Ellipticine can also act as an inhibitor or inducer of biotransformation enzymes, thereby modulating its own metabolism leading to its genotoxic and pharmacological effects. The toxicity of ellipticine to U87MG glioblastoma cells and mechanisms of its action to these cells are aims of this study.r />r /> METHODS: Ellipticine metabolites formed in U87MG cells were analyzed using HPLC. Covalent DNA modifications by ellipticine were detected by 32P-postlabeling. CYP enzyme expression was examined by QPCR and Western blot.r />r /> RESULTS: U87MG glioblastoma cell proliferation was efficiently inhibited by ellipticine. This effect might be associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by 13-hydroxy- and 12-hydroxyellipticine, the ellipticine metabolites generated by CYP1A1, 1B1 and 3A4, lactoperoxidase and cyclooxygenase 1, the enzymes expressed in U87MG cells. Moreover, by inducing CYP1B1, 3A4 and 1A1 enzymes in U87MG cells, ellipticine increases its own enzymatic activation, thereby enhancing its own genotoxic and pharmacological potential in these cells. Ellipticine concentration used for U87MG cell treatment is extremely important for its pharmacological effects, as its metabolite profiles differed substantially predicting ellipticine to be either detoxified or activated.r />r /> CONCLUSION: The results found in this study are the first report showing cytotoxicity and DNA adduct formation by ellipticine in glioblastomas....

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Journal Article 2009; 30(Suppl 1): 52-59 PubMed PMID: 20027145 Citation  Keywords:  Animals, Benz(a)Anthracenes:metabolism, Chromatography, High Pressure Liquid:methods, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System:metabolism, Humans, Inhibitory Concentration 50, Kinetics, Male, Microsomes, Liver:enzymology, Oxidati.   

OBJECTIVES: 3-Aminobenzanthrone (3-ABA) is the main human metabolite of carcinogenic environmental pollutant 3-nitrobenzanthrone (3-NBA). Understanding which cytochrome P450 (CYP) enzymes are involved in metabolism of this toxicant is important in the assessment of individual susceptibility. Characterization of 3-ABA metabolites formed by rat hepatic microsomes containing cytochromes P450 (CYPs) and identification of the major rat and human CYPs participating in this process are aims of this study.r />r /> METHODS: HPLC with UV detection was employed for the separation and characterization of 3-ABA metabolites. Inducers and inhibitors of CYPs and rat and human recombinant CYPs were used to characterize the enzymes participating in 3-ABA oxidation.r />r /> RESULTS: Selective CYP inhibitors and hepatic microsomes of rats pre-treated with specific CYP inducers were used to characterize rat liver CYPs metabolizing 3-ABA (measured as consumption of 3-ABA). Kinetics of these reactions catalyzed by rat hepatic microsomes was also evaluated. Based on these studies, we attribute most of 3-ABA metabolism in rat liver to CYP1A and 3A. Among recombinant rat and human CYP enzymes tested in this study, rat CYP3A2 and human CYP3A4/5, followed by CYP1A1 of both organisms were the most effective enzymes converting 3-ABA. Rat hepatic CYP enzymes oxidize 3-ABA up to three metabolites. Two of them were identified to be the products formed by oxidation of 3-ABA on its amino group back to the parent compound from which 3-ABA is generated in organisms, 3-NBA. Namely, N-hydroxylation metabolite, N-hydroxy-3-ABA and 3-NBA were identified to be these 3-ABA oxidation products. These metabolites are formed by CYPs of a 1A subfamily. Another 3-ABA metabolite, whose structure remains to be characterized, is generated not only by CYP1A but also by other CYP enzymes, predominantly by CYPs of a 3A subfamily.r />r /> CONCLUSION: The results found in this study, the first report on the metabolism of 3-ABA by human and rat CYPs, clearly demonstrate that CYPs of 3A and 1A subfamilies are the major enzymes metabolizing 3-ABA....

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Journal Article 2009; 30(Suppl 1): 46-51 PubMed PMID: 20027144 Citation  Keywords:  Animals, Anisoles:metabolism, Chromatography, High Pressure Liquid:methods, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System:metabolism, Humans, Inhibitory Concentration 50, Kinetics, Microsomes, Liver:enzymology, Nitrophenols:metabolism.   

OBJECTIVES: 2-Nitrophenol (2-NP) is the major detoxification metabolite of an important industrial pollutant and a potent carcinogen, 2-nitroanisole (2-NA). Characterization of the products of 2-NP metabolism by rat hepatic microsomes containing cytochromes P450 (CYPs) and identification of the major CYP enzymes participating in this process are aims of this study.r />r /> METHODS: HPLC with UV detection was employed for the separation and characterization of 2-NP metabolites. Inducers and inhibitors of CYPs and rat recombinant CYPs were used to characterize the enzymes participating in 2-NP oxidation.r />r /> RESULTS: Rat hepatic microsomes oxidize 2-NP to its hydroxylated metabolite, 2,5-dihydroxynitrobenzene (2,5-DNB). No nitroreductive metabolism leading to the formation of o-aminophenol was evident when using rat hepatic microsomes. Selective CYP inhibitors and hepatic microsomes of rats pre-treated with specific CYP inducers were used to characterize CYPs oxidizing 2-NP in rat livers. Based on these studies, we attribute most of 2-NP oxidation in rat liver to CYP2E1 and 3A, followed by CYP2D and 2C. Among recombinant rat CYP enzymes tested in this study, CYP2E1 and 2C11 were the most effective enzymes oxidizing 2-NP. Oxidation of 2-NP by rat CYP2E1 exhibits the Michaelis-Menten kinetics, having the Km value of 0.35 mM.r />r /> CONCLUSION: The results found in this study, the first report on the metabolism of 2-NP by rat hepatic microsomes and rat CYP enzymes, demonstrate that CYP2E1 is the major enzyme oxidizing this compound in rat liver....

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Journal Article 2009; 30(Suppl 1): 41-45 PubMed PMID: 20027143 Citation  Keywords:  Animals, Blotting, Western, Cytochrome P-450 Enzyme System:metabolism, Female, Lipopolysaccharides:metabolism, Liver:enzymology, Probiotics:metabolism, Rats, Rats, Wistar, Time Factors,.   

OBJECTIVES: The aim of the study was to find, whether probiotic Escherichia coli Nissle 1917 O6:K5:H1 (EcN) influence the amount and activity of cytochromes P450 (CYP) in rat liver.r />r /> DESIGN: Live bacterial suspension of EcN was applied to the female Wistar rats in single dose or for 14 days consecutively. The bacterial lipopolysaccharide (LPS) isolated by phenol extraction from the EcN was given to the rats for 14 days as well. Control rats were treated with the saline solution daily for 14 days. Relative amount of CYP2C6, CYP2C9 (corresponding to rat CYP2C11), CYP3A1 and CYP1A2 protein expression in rat liver microsomes was determined by Western blotting. For the determination of six CYP activities (corresponding to human CYP1A2, CYP2A6, CYP2B6, CYP3A4, CYP2C9 and CYP2D6) fluorescence, luminescence or absorbance detection was used.r />r /> RESULTS: The data presented show that the changes of the total content of the CYP enzymes in rat liver are not significant after administration of the probiotic for 1 or 14 days as well as of the LPS. Western blots revealed a slight increase in CYP2C6 protein expression; level of another rat CYP2C protein (readings with anti-human CYP2C9 antibody corresponding to the rat CYP2C11) as well as of CYP1A2 was elevated after administration of LPS; a small decrease was observed with CYP3A1 protein. Changes in activities of CYP forms are not significant, only the activity of rat CYP2C forms in liver microsomal samples of rats given free LPS appeared to exhibit a small, but significant tendency to increase.r />r /> CONCLUSION: The results show that the p.o. administration of probiotics to rat does not markedly influence the rat hepatic CYP enzymes....

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Journal Article 2009; 30(Suppl 1): 36-40 PubMed PMID: 20027142 Citation  Keywords:  Animals, Cytochrome P-450 CYP1A2:metabolism, Cytochrome P-450 CYP1A2 Inhibitors, Cytochrome P-450 CYP2E1:metabolism, Cytochrome P-450 CYP2E1 Inhibitors, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System:metabolism, Humans, Indoles:metabol.   

OBJECTIVES: To study the contribution of individual purified porcine CYP1A2, 2E1 and 2A19 enzymes to the biotransformation of skatole.r />r /> METHODS: Individual porcine and human enzymes (CYP1A2, 2E1 or 2A6/19) were used to study their potential involvement in skatole metabolism. Furthermore, the inhibition experiments using specific inhibitors of CYP1A2, 2E1 or 2A6/19, were performed. For determination of skatole biotransformation by individual CYP forms in reconstituted systems, HPLC method with UV detection was used.r />r /> RESULTS: The data presented in this paper show that porcine and human CYPs are responsible for the formation of indole-3-carbinol and 3-methyloxindole. Whereas in pig CYP2A19 and CYP1A2 seem to be the most important for metabolism of skatole, in man CYP1A2 and CYP2E1 forms are mainly responsible for the production of the metabolites mentioned above.r />r /> CONCLUSIONS: The porcine and human CYP1A2, 2E1, 2A6/19 forms contribute to formation of 3-methyloxindole and indole-3-carbinol....

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Journal Article 2009; 30(Suppl 1): 32-35 PubMed PMID: 20027141 Citation  Keywords:  Antioxidants:metabolism, Cardiovascular Diseases:metabolism, Humans, Hypertension:metabolism,.   

OBJECTIVES: This paper reviews and compares major approaches and strategies to modulation of antioxidative response in the therapy of hypertension and cardiovascular diseases.r />r /> DESIGN: There are two major strategies of modulation of antioxidative response in hypertension and cardiovascular diseases: (i) modulation of NO levels by NOS stimulation, increase of NO bioavailability, administration of NO, and NOS gene incorporation; (ii) scavenging of superoxide and suppression of oxidative stress by activation of antioxidant gene expression or by suppression of selected genes by RNA silencing. These strategies are accomplished by several concepts, including (1) delivery of external agents, (2) antioxidant gene therapy and RNA silencing, and (3) combined therapies and approaches.r />r /> CONCLUSION: Combined therapies and approches often achieve multiplicative effects and are the most promising attitude in antioxidant-oriented therapy of hypertension and cardiovascular diseases....

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Case Reports 2009; 30(Suppl 1): 29-31 PubMed PMID: 20027140 Citation  Keywords:  Diagnosis, Differential, Humans, Hyperbilirubinemia, Neonatal:diagnosis, Infant, Newborn, Male, Mevalonate Kinase Deficiency:complications,.   

OBJECTIVES: To present clinical and laboratory findings in the case of a term newborn with conjugated hyperbilirubinaemia and to stress the importance of differential diagnosis.r />r /> RESULTS: A term newborn delivered by caesarean section (birth weight 2550 g, birth length 47 cm, value of Apgar score 9/10) with good direct adaptation had on the first day of life increased levels of conjugated bilirubin (23 micromol/l), unconjugated bilirubin (55 micromol/l) and C-reactive protein 39.4 g/l. The diagnosis of mevalonic aciduria was confirmed by urine analysis (mevalonolactone 393 micromol/mmol crea, normal range <2.0 micromol/mmol crea; mevalonic acid 40.5 micromol/mmol crea, normal range <0.04 micromol/mmol crea).r />r /> CONCLUSION: Mevalonic aciduria can be clinically distinguished based on symptoms of neurological involvement. It can also present itself with hepatosplenomegaly, lymphadenopathy, anaemia, leukocytosis, increased sedimentation rates and levels of C-reactive protein. In cases of conjugated hyperbilirubinaemia of unknown aetiology it is important to exclude mevalonic aciduria by urine investigation for organic acids....

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Journal Article 2009; 30(Suppl 1): 25-28 PubMed PMID: 20027139 Citation  Keywords:  Adenomatous Polyposis Coli:genetics, Cohort Studies, Exons, Family, Genes, APC, Genetic Predisposition to Disease, Humans, Mutation, Polymorphism, Genetic, Polymorphism, Single-Stranded Conformational, Sequence Analysis, DNA, Slovakia,.   

OBJECTIVES: Polymorphism in the adenomatous polyposis coli (APC) gene was analyzed in 33 families suspected of familial adenomatous polyposis (FAP) without identified APC gene mutation. Screening of 104 members of mentioned families for polymorphism in the APC gene, was performed using single strand conformation polymorphism (SSCP) and DNA sequencing.r />r /> RESULTS: Twelve different types of polymorphism were found in the cohort of the families analyzed. Nine polymorphisms were located within exon 15, one within exon 6, one within exon 11 and one within exon 13. Of the 12 polymorphisms, 11 were silent substitution and only one was responsible for the amino acid change - D1822V, which was identified in 60% of the families analyzed.r />r /> CONCLUSION: The most frequently detected polymorphism D1822V is potentially associated with the risk of colorectal cancer. Three detected polymorphisms - Y486Y, T1493T and S1756S - also seem to be associated with colon cancer risk. All these polymorphisms may be used as markers for diagnosis of colorectal cancer. The importance of other detected polymorphisms remains still unclear, but their involvement is being continuously observed....

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