Daniel Francisco Arencibia-Arrebola, Luis Alfredo Rosario-Fernández, Yolanda Emilia Suárez-Fernández, Alexis Vidal-Novoa and Livan Delgado-Roche
The aim of this research was to compare the efficiency of different murine lines for genotoxicity assays. Rats and mice of different murine lines were used. The spontaneous and induced indexes were evaluated according to alkaline comet assay of peripheral blood leukocytes, micronucleus and chromosomic aberration assay of bone marrow cell, and sperm head morphology assay. In most of the evaluated assays the line of Balb/c mice turned out to be the ideal biomodel, with less spontaneous indexes and high induced indexes to the mutagen used; allowing to detect in a narrow error margin those substances that are classified of very low genotoxicity. These results demonstrate that genetically the line of Balb/c mice in both sexes is more stable than the other ones evaluated. This suggests the use of the Balb/c line on in vivo genotoxicity assay will increase sensibility and robustness.
Ivana Milackova, Lucia Kovacikova, Miroslav Veverka, Ján Gallovic and Milan Stefek
The group of 21 novel semi-synthetic derivatives of quercetin was screened for the antiradical efficiency in a DPPH assay. The initial fast absorbance decrease of DPPH, corresponding to the transfer of the most labile H atoms, was followed by a much slower absorbance decline representing the residual antiradical activity of the antioxidant degradation products. Initial velocity of DPPH decolorization determined for the first 75-s interval was used as a marker of the antiradical activity. Application of the kinetic parameter allowed good discrimination between the polyphenolic compounds studied. The most efficient chloronaphthoquinone derivative (compound Ia) was characterized by antiradical activity higher than that of quercetin and comparable with that of trolox. Under the experimental conditions used, one molecule of Ia was found to quench 2.6±0.1 DPPH radicals.
Soňa Fraňová, Anna Strapková, Juraj Mokrý, Martina Šutovská, Marta Jošková, Vladimíra Sadloňová, Martina Antošová, Darina Pavelčíková, Dana Flešková and Gabriela Nosáľová
muscle contraction during allergen-induced hyperreactivity of the airways. J Pharm Pharmacol 59 : 727-732.
Franova S, Joskova M, Novakova E, Adamicova K, Sutovska M, Nosal S. (2009) Effects of flavin7 on allergen induced hyperreactivity of airways. Eur J Med Res 7 : 78-81.
Franova S, Joskova M, Sutovska M, Novakova E, Pechanova O, Nosalova G. (2010) The efficiency of polyphenolic compounds on allergen induced hyperreactivity of the airways. Biomedicine & Pharmacotherapy doi:10.1016/j. biopha.2010
Marie Stiborová, Jitka Poljaková, Eva Martínková, Lucie Bořek-Dohalská, Tomáš Eckschlager, Rene Kizek and Eva Frei
-mediated oxidation of anticancer alkaloid ellipticine dictates its antitumor efficiency. Biochim Biophys Acta 1814 : 175-185.
Sugikawa E, Hosoi T, Yazaki N, Gamanuma N, Nakanishi N and Ohashi M. (1999). Mutant p53 mediated induction of cell cycle arrest and apoptosis at G1 phase by 9-hydroxyellipticine. Anticancer Res 19 : 3099-3108.
Marie Stiborová, Jitka Poljaková, Tomáš Eckschlager, Rene Kizek and Eva Frei
, Moserová M, Hudeček J, Phillips DH and Frei E. (2008). Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: studies with the hepatic NADPH: cytochrome P450 reductase null mouse. Toxicol Appl Pharmacol 226 : 318-327.
Stiborová M, Rupertová M and Frei E. (2010). Cytochrome P450- and peroxidase-mediated oxidation of anticancer alkaloid ellipticine dictates its antitumor efficiency. Biochim Biophys Acta , in press.
Schwab M. (1999). Human neuroblastoma: from basic science to clinical
Martina Svobodová, Helena Dračínská, Markéta Martínková, Jiří Hudeček, Petr Hodek, Eva Frei and Marie Stiborová
Oxidation of carcinogenic 2-nitroanisole by rat cytochromes P450 - similarity between human and rat enzymes
2-Nitroanisole (2-NA) is an important industrial pollutant and a potent carcinogen for rodents. Understanding which cytochrome P450 (CYP) enzymes are involved in its metabolism are important to assess an individual's susceptibility to this environmental carcinogen. The aim of this study was to evaluate the efficiency of rat hepatic CYPs to oxidize 2-NA, to examine the metabolites formed during such an oxidation, and to compare such efficiencies of rat CYPs with those of human. 2-NA is oxidized by rat hepatic microsomes to 2-nitrophenol (2-NP) as the major metabolite, and to 2,6-dihydroxynitrobenzene (2,6-DNB) and 2,5-dihydroxynitrobenzene (2,5-DNB) as the minor products. All these metabolites are suggested as detoxication products. Using hepatic microsomes of rats pre-treated with specific CYP inducers and microsomes from Baculovirus transfected insect cells expressing recombinant rat and human CYP enzymes we found that rat recombinant CYP2E1, 2D2, 2B2, 2C6 and 1A1, as well as orthologous human CYP enzymes are the most efficient enzymes metabolizing 2-NA. However, human CYP1A1 oxidize 2-NA with a higher efficiency than the enzyme of rats. The results show the participation of orthologous CYPs in 2-NA oxidation by both species and underline the suitability of rat species as a model to evaluate human susceptibility to 2-NA.
Lenka Vilímková, Jan Páca, Veronika Kremláčková, Jan Páca and Marie Stiborová
Isolation of cytoplasmic NADPH-dependent phenol hydroxylase and catechol-1,2-dioxygenase from Candida tropicalis yeast
The efficiencies of NADPH-dependent phenol hydroxylase (EC 18.104.22.168) and catechol 1,2-dioxygenase (EC.22.214.171.124) in biodegradation of phenol in the cytosolic fraction isolated from yeast Candida tropicalis were investigated. Enzymatic activities of both NADPH-dependent phenol hydroxylase and catechol 1,2-dioxygenase were detected in the cytosolic fraction of C. tropicalis grown on medium containing phenol. Using the procedure consisting of chromatography on DEAE-Sepharose, fractionation by polyethylene glycol 6000 and gel permeation chromatography on Sepharose 4B the enzyme responsible for phenol hydroxylation in cytosol, NADPH-dependent phenol hydroxylase, was isolated from the cytosolic fraction of C. tropicalis close to homogeneity. However, fractionation with polyethylene glycol 6000 lead to a decrease in catechol 1,2-dioxygenase activity. Therefore, another procedure was tested to purify this enzyme. Gel permeation chromatography of proteins of the eluate obtained by chromatography on a DEAE-Sepharose column was utilized to separate phenol hydroxylase and catechol 1,2-dioxygenase. Among gel permeation chromatography on columns of Sephadex G-100, Sephacryl S-300 and Sepharose 4B tested for their efficiencies to isolate phenol hydroxylase and catechol 1,2-dioxygenase, that on Sephacryl S-300 was found to be suitable for such a procedure. Nevertheless, even this chromatographic method did not lead to obtain catechol 1,2-dioxygenase in sufficient amounts and purity for its further characterization. The data demonstrate the progress in resolving the enzymes responsible for the first two steps of phenol degradation by the C. tropicalis strain.
Rami B. Kassab, Ondrej Vasicek, Milan Ciz, Antonin Lojek and Tomas Perecko
The health benefits of berberine have been recognized for years. Even so, its effects on human neutrophils, the first line of immune defense, have not been reported. The purpose of this study was to investigate the effects of berberine on the human neutrophil oxidative burst. Reactive oxygen species production was analyzed by luminol-enhanced chemiluminescence. The analysis was performed in spontaneous and stimulated (phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP)) whole blood and isolated neutrophils in the presence or absence of berberine. The effects of berberine on oxidant production in cell-free assays were evaluated using luminescence (H2O2-peroxidase-luminol) and fluorescence (Oxygen Radical Absorbance Capacity – ORAC) techniques. Berberine decreased the production of reactive oxygen species in human whole blood and isolated neutrophils stimulated with either PMA or OZP with a different efficiency (EC50 was 69 μM and 197 μM for PMA and OZP, respectively). The effect was more pronounced in isolated neutrophils. Cell-free assays showed the antioxidant activity of berberine against peroxyl radicals and hydrogen peroxide. Based on our results, we suggest that the effects of berberine on reactive oxygen species production in human neutrophils are due to its antioxidant activity.
Jana Mizerovská, Helena Dračínská, Volker Arlt, Jiří Hudeček, Petr Hodek, Heinz Schmeiser, Eva Frei and Marie Stiborová
Rat cytochromes P450 oxidize 3-aminobenzanthrone, a human metabolite of the carcinogenic environmental pollutant 3-nitrobenzanthrone
3-Aminobenzanthrone (3-ABA) is a human metabolite of carcinogenic 3-nitrobenzanthrone (3-NBA), which occurs in diesel exhaust and air pollution. Understanding which cytochrome P450 (CYP) enzymes are involved in metabolic activation and/or detoxication of this toxicant is important in the assessment of an individual's susceptibility to this substance. The aim of this study was to evaluate the efficiency of rat hepatic CYPs to oxidize 3-ABA and to examine the metabolites formed during such an oxidation. The metabolites formed by CYPs in rat hepatic microsomes were separated by high performance liquid chromatography (HPLC). 3-ABA is oxidized by these enzymes to three metabolites, which were separated by HPLC as distinguish product peaks. Using co-chromatography with synthetic standards, two of them were identified to be oxidative metabolites of 3-ABA, N-hydroxy-3-ABA and 3-NBA. The structure of another 3-ABA metabolite remains to be characterized. To define the role of rat hepatic CYP enzymes in metabolism of 3-ABA, we investigated the modulation of its oxidation using different inducers of CYPs for treatment of rats to enrich the liver microsomes with individual CYPs. Based on these studies, we attribute most of 3-ABA oxidation in rat hepatic microsomes to CYP2B, followed by CYP1A, although a role of other hepatic CYPs cannot be ruled out. Inhibition of 3-ABA oxidation by selective inhibitors of individual CYPs, supported this finding.
Guava (Psidium guajava L.) is a fruit tree largely used in folk medicine in tropical and subtropical areas. This exotic species was introduced in a botanical garden in the northeast of Algeria in the 1950’s. The aim of this study is to estimate, for the first time, the antioxidant and anticholinesterase activities of chloroform, ethyl acetate and n-butanol extracts of P. guajava growing in Algeria. Six antioxidant assays were tested, results showed very important efficiency in free radical scavenging, reducing power and β-carotene bleaching of tested extracts. Values of IC50 or A0.5 of some samples were lower than those of standards. With regard to anticholinesterase activity, the inhibitory of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was investigated. The extracts exhibited interesting capacity to inhibit these enzymes with low values of IC50 and even less than that of galanthamine. These activities were correlated with total phenolic content which was more important compared to the one found in extracts from trees growing in tropical and subtropical region. This could be due to resistance and adaptation of P. guajava grown in Algeria. The data obtained suggest the use of bioactive compounds from P. guajava leaves as antioxidant and drugs for symptomatic treatment of Alzheimer disease.