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Comparative Study 2009; 30(Suppl 1): 133-136 PubMed PMID: 20027159 Citation  Keywords:  Benzothiepins:pharmacology, Brompheniramine:pharmacology, Chlorpheniramine:pharmacology, Dose-Response Relationship, Drug, Extracellular Space:drug effects, Histamine H1 Antagonists:administration & dosage, Humans, Intracellular Space:drug effects, Lorata.   

OBJECTIVES: We analysed and compared the effect of five H1-antihistamines on stimulated oxidative burst at extra- and intracellular level of isolated and stimulated human polymorphonuclear leukocytes.

DESIGN: Oxidative burst of isolated human neutrophils was studied by means of luminol and isoluminol enhanced chemiluminescence.

RESULTS: The following rank order of potency for H1-antihistamines to decrease chemiluminescence was evaluated extracellularly: dithiaden> loratadine> chlorpheniramine> brompheniramine> pheniramine and at intracellular site: loratadine> dithiaden.

CONCLUSION: H1-antihistamines differ substantially according to their chemical structure in suppressing oxidative burst both at extra- and intracellular site of isolated stimulated human neutrophils....

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Journal Article 2009; 30(Suppl 1): 128-132 PubMed PMID: 20027158 Citation  Keywords:  Absorption, Animals, Cattle, Centrifugation, Collagen Type I:chemistry, Electrophoresis, Polyacrylamide Gel, Humans, Hydrogen Peroxide:chemistry, Hydroxyl Radical:chemistry, Luminescence, Oxidation-Reduction, Phagocytes:metabolism, Platelet Aggregation, P.   

OBJECTIVES: The effects of non-modified and oxidatively modified calf skin collagen type I on platelet aggregation and the oxidative burst of phagocytes were examined in the framework of a general hypothesis that collagen, platelets and phagocytes cooperate to modulate the oxidative burst of phagocytes and the extent of oxidative stress.

MATERIALS AND METHODS: Calf skin collagen type I was subjected to oxidative modification by hydrogen peroxide or hydroxyl radical. Thermal denaturation of collagen was performed in a spectrophotometer equipped with a temperature gradient device. The aggregation of isolated human platelets obtained after differential centrifugation was measured using a dual-channel aggregometer. The production of reactive oxygen species by human whole blood phagocytes was evaluated by luminol-enhanced chemiluminescence.

RESULTS: Oxidative modification of collagen samples was characterized by a decrease in denaturation transition temperature. Oxidatively modified samples showed a modified SDS-PAGE pattern, evidencing a significant destruction of the collagen. All oxidatively modified collagen samples, independent of the oxidation treatment applied, lost their platelet-aggregating and phagocyte oxidative burst-inducing activity.

CONCLUSION: The results suggest that reactive oxygen species were able to modify collagen. On the other hand, oxidatively modified collagen lost its activating properties towards platelets and phagocytes....

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Comparative Study 2009; 30(Suppl 1): 121-127 PubMed PMID: 20027157 Citation  Keywords:  3T3 Cells, Adenosine Triphosphate:metabolism, Animals, Cell Count:methods, Cell Line, Cell Proliferation, Cells, Cultured, Colorimetry, Dermis:cytology, Electric Impedance, Fibroblasts:cytology, Humans, Keratinocytes:cytology, Luminescent Measurements, Mi.   

OBJECTIVES: Determination of cell numbers is a crucial step in studies focused on cytokinetics and cell toxicity. The impedance-based analysis employing electronic sensor array system xCELLigence System allowing label-free dynamic monitoring of relative viable adherent cell amounts was compared with the most utilized methods for relative quantification of viable cell numbers based on a determination of cellular metabolism.

DESIGN: Colorimetric assay based on reduction of tetrazolium salt (MTT) by mitochondrial enzymes and chemiluminiscent assay based on intracellular adenosine triphosphate (ATP) determination were compared with the impedance-based system. Cell morphology was compared by microscopic evaluation. Normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF), together with 3T3 mouse fibroblast and HaCaT keratinocyte cell lines were employed.

RESULTS: The progress of cell growth curves obtained by different methods during 72 hours reflected cell type and cell seeding densities. The impedance-based method was found to be applicable for the determination of the cell proliferation of 3T3 fibroblasts, HaCaT and NHDF, since the comparison of this method with ATP and MTT determinations showed a comparable results. In contrast, the proliferation of NHEK measured by the impedance-based method did not correlate with other methodological approaches. This could be accounted to the specific morphological appearance of these cells.

CONCLUSION: The study shows the impedance-based detection of viable adherent cells is a valuable approach for cytokinetics and pharmacological studies. However, the specific morphological characteristics of cell lines have to be considered employing this method for determination of cell proliferation without using other reference methods....

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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): 101-105 PubMed PMID: 20027153 Citation  Keywords:  Biological Transport:drug effects, Caco-2 Cells, Caffeine:pharmacokinetics, Cell Culture Techniques, Cell Survival:drug effects, Diffusion, Humans, Hydrogen-Ion Concentration, Intestinal Absorption, Intestinal Mucosa:metabolism, Kinetics, Linear Models, M.   

OBJECTIVES: The Caco-2 cell monolayer model is widely used as a standard screening tool for studying the mechanisms of cellular drug transport. Caffeine was chosen as a model drug and is supposed to be class I of the Biopharmaceutics Classification System (BCS). Our study was conducted 1) to characterize the mechanisms of caffeine transport across the intestinal barrier, 2) to classify caffeine according to BCS, 3) to predict drugs intestinal absorption in humans.

METHODS: Caffeine transport (0.1, 0.3, 1 and 10 mmol/l) was studied in Caco-2 cell monolayer in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under iso-pH 7.4 and pH-gradient (6/7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+- free transport medium (opening tight junctions).

RESULTS: The caffeine transport was linear with time, transport direction and pH independent, displaying non-saturable (first-order) kinetics, with high permeability coefficient (Papp): in AP-BL direction Papp = 46.3-53.5 x 10-6 cm/s; in BL-AP direction Papp = 45.6-49.4 x 10-6 cm/s. Thus, the transport seems to be transcellular mediated by passive diffusion. Using Ca2+- free transport medium tight junctions were opened (confirmed by increased Papp of mannitol) but the caffeine Papp was not changed. Thus, the paracellular route is only a minor way of caffeine transport.

CONCLUSION: High solubility and high permeability of caffeine rank it among class I of BCS and well absorbed compounds....

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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(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.

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.

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.

CONCLUSIONS: The porcine and human CYP1A2, 2E1, 2A6/19 forms contribute to formation of 3-methyloxindole and indole-3-carbinol....

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