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Journal Article 2001; 22(1): 53-73 PubMed PMID: 11393163 Citation  Keywords:  Adrenal Glands:physiology, Animals, Biological Clocks, Blood Pressure:physiology, Chronobiology Disorders:physiopathology, Chronobiology Phenomena, Chronotherapy, Circadian Rhythm:physiology, Drug Administration Schedule, Genomics, Homeostasis, Humans, Pi.   

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Journal Article 2009; 30(Suppl 1): 116-120 PubMed PMID: 20027156 Citation  Keywords:  Adult, Aldehydes:blood, Antioxidants:metabolism, C-Reactive Protein:metabolism, Cholesterol, HDL:blood, Chromatography, High Pressure Liquid, Female, Humans, Luminescent Measurements, Male, Malondialdehyde:blood, Middle Aged, Nitrites:blood, Oxidative Str.   

OBJECTIVES: Chronic pancreatitis (CP) is a heterogeneous disease defined as chronic inflammatory changes of the pancreatic tissue caused by variety of aetiologies. Oxidative stress accompanying the inflammatory processes has been suggested as an important factor contributing to CP development. The aim of this study was to determine levels of lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), together with nitrites and the total antioxidant capacity in the plasma of patients with CP and control subjects.

DESIGN: One hundred and five patients with chronic pancreatitis and twenty seven healthy controls were included into this study. Levels of MDA and 4-HNE were analyzed using high-performance liquid chromatography. The total antioxidant capacity of plasma against peroxyl radicals was evaluated using chemiluminescent determination. Nitrites were determined using Griess reaction. Biochemical and haematological parameters were measured by standard methods.

RESULTS: The plasma levels of both MDA and 4-HNE, together with the plasma levels of nitrites, were significantly higher in CP patients, compared to healthy controls. The total antioxidant capacity did not differ significantly. Biochemical parameters were in the normal range. The MDA and 4-HNE levels correlated positively with the levels of high-density lipoprotein cholesterol. Nitrite levels correlated positively with C-reactive protein, total white blood cells, and triglycerides.

CONCLUSION: The significantly increased plasma levels of MDA, 4-HNE, and nitrites indicate that oxidative stress is present in patients with CP and that it may play a role in initiation and maintenance of inflammation within the pancreatic tissue in CP patients....

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Journal Article 2009; 30(Suppl 1): 80-87 PubMed PMID: 20027149 Citation  Keywords:  Candida tropicalis:enzymology, Catechol 1,2-Dioxygenase:chemistry, Catechols:chemistry, Chromatography, Chromatography, Gel, Chromatography, High Pressure Liquid, Cytosol:chemistry, Dextrans, Dimerization, Electrophoresis, Polyacrylamide Gel, Fungal Prote.   

OBJECTIVES: Candida tropicalis yeast is a microorganism that possesses high tolerance for phenol and shows strong phenol degrading activity. This yeast is capable of utilizing phenol as the sole carbon and energy source. While the enzyme participating on the first step of phenol biodegradation, NADPH-dependent phenol hydroxylase, has already been characterized, information on the enzyme participating in the second step of its degradation, catechol 1,2-dioxygenase, is scarce. The development of the procedure suitable for catechol 1,2-dioxygenase isolation and partial characterization of this enzyme are the aims of this study.

METHODS: Combination of chromatography on DEAE-Sepharose and gel-permeation chromatography on Sephadex G-100 was used for isolation of cytosolic catechol 1,2-dioxygenase from C. tropicalis yeast. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography on Sephadex G-100 were used to evaluate the molecular mass of the enzyme. The enzyme activity was followed by HPLC (catechol consumption and/or cis,cis-muconic acid formation).

RESULTS: Using the isolation procedure consisting of chromatography and re-chromatography on a column of DEAE-Sepharose and gel filtration on Sephadex G-100, catechol 1,2-dioxygenase was purified from C. tropicalis cytosol to homogeneity. Catechol 1,2-dioxygenase was found to be a homodimer with a subunit molecular mass of 30000 +/- 5000. The enzyme oxidized catechol producing cis,cis-muconic acid. The optimal temperature and pH were 30 degrees C and 7.7, respectively.

CONCLUSIONS: The data are the first report showing the isolation of eukaryotic catechol 1,2-dioxygenase from C. tropicalis to homogeneity and its partial characterization....

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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.

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.

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.

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.

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.

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.

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.

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.

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.

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(4): 491-495 PubMed PMID: 20010507 Citation  Keywords:  Adult, Autonomic Nervous System:physiology, Baroreflex:physiology, Blood Pressure:physiology, Diabetes Mellitus, Type 1:physiopathology, Diabetic Neuropathies:physiopathology, Female, Humans, Hyperglycemia:physiopathology, Linear Models, Male,.   

OBJECTIVES: To date, the clinical usefulness of measuring baroreflex sensitivity (BRS) to detect impairment of the autonomic nervous system in patients with diabetes mellitus (DM) type I has not been evaluated sufficiently (Mlcáková et al. 2008). The aim of the current study was the determination and statistical comparison of the mean values of BRS in our DM type I patients cohort and in a control group of healthy volunteers as well as the determination of BRS value dependency on the duration of diabetes and the level of glycemic control in DM I patients. We also aimed to determine the inter-individual and intra-individual variability of BRS in our patients.

MATERIAL AND METHODS: We examined 100 patients with type I diabetes mellitus (37 women and 63 men, mean age 30 years, duration of the disease >or= 10 years) and 40 healthy, age- and sex-matched, subjects. Data from the patient cohort were subsequently analysed for duration of the diabetes and the level of glycemic control as assessed by glycated haemoglobin (HbA1c). We used a simple proportional test to compare the occurrence of impaired BRS in the patient cohort and the control group, and a simple linear regression to assess associations between BRS and duration of the diabetes and the levels of glycemic control.

RESULTS: The mean BRS value in our group of diabetic patients and the control group were 10.15 ms/mmHg and 13.35 ms/mmHg, respectively. II. Statistically significant association between BRS impairment and the duration of the disease or level of glycemic control was not confirmed in our patient cohort. III. We observed an increased inter-individual variability and a relatively low intra-individual variability of BRS in patients with DM type I.

CONCLUSIONS: We found a statistically highly significant difference between the proportions of impaired BRS in the group of diabetics vs. control. However, BRS did not correlate with the duration of the disease or with the level of glycemic control significantly. Albeit not reaching statistical significance, trends could be observed, which we consider clinically interesting....

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Journal Article 2009; 30(3): 352-356 PubMed PMID: 19855358 Citation  Keywords:  Acetic Acid:toxicity, Analgesia, Analysis of Variance, Animals, Anti-Inflammatory Agents, Non-Steroidal:pharmacology, Celecoxib, Chromatography, High Pressure Liquid, Dose-Response Relationship, Drug, Drug Synergism, Expectorants:administration & dosage, .   

OBJECTIVES: Previously, we found that guaifenesin enhances analgesia induced by paracetamol. The aim of the present study was to determine whether guaifenesin is able to also increase analgesic activity in the non-steroid anti-inflammatory drugs ibuprofen, nimesulide and celecoxib. In addition we investigated the influence of guaifenesin on plasma levels of nimesulide.

METHODS: A model of visceral pain consisting of intraperitoneal injection of acetic acid (writhing test) was used. Levels of nimesulide in plasma were measured by HPLC. All drugs were given orally and tested in mice.

RESULTS: Guaifenesin alone did not produce any antinociceptive effect. Simultaneous administration of guaifenesin (200 mg/kg) and subanalgesic doses of ibuprofen (10 and 30 mg/kg), nimesulide (10 and 20 mg/kg) or celecoxib (1 and 5 mg/kg) resulted in a significant antinociceptive effects. The plasma levels of nimesulide were significantly higher in combination with guaifenesin at 30, 60 and 90 min after oral administration in comparison to nimesulide monotherapy.

CONCLUSION: The present results suggest that guaifenesin might enhance the analgesic activity of various non-steroidal anti-inflammatory drugs....

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