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Evaluation Studies 2006; 27(Suppl 2): 44-47 PubMed PMID: 17159777 Citation  Keywords:  Caco-2 Cells, Cells, Cultured, Cytotoxins:pharmacology, DNA:drug effects, DNA Damage, Humans, Monoterpenes:pharmacology, Mutagenicity Tests, Plant Oils:pharmacology, Protective Agents:pharmacology, Thymol:pharmacology,.   

OBJECTIVES: To investigate cytotoxic, genotoxic and DNA-protective effects of carvacrol and thymol on human hepatoma HepG2 and colonic Caco-2 cells cultured in vitro.

METHODS AND RESULTS: Cytotoxicity testing was performed by the trypan blue exclusion technique. Level of DNA lesions induced in human cells with carvacrol, thymol or their combinations with hydrogen peroxide (H(2)O(2)) were measured by alkaline single cell gel electrophoresis (comet assay). The trypan blue exclusion technique showed that though the metabolically more competent human hepatoma HepG2 cells were more sensitive to the toxic effects of carvacrol or thymol than colonic Caco-2 cells, which contained lower levels of metabolizing enzymes, the observed differences were not very expressive. The comet assay technique showed that at concentrations <IC(50) the compounds studied did not induce DNA strand breaks either in hepatoma HepG2 or in colonic Caco-2 cells. On the other side, 24-h incubation of cells in the presence of carvacrol or thymol led to a significant protection of the cells studied toward DNA damage induced by H(2)O(2).

CONCLUSIONS: Based on our results it can be concluded that the cytotoxic effects of selected components of essential plant oils on human hepatoma HepG2 and colonic Caco-2 cells were not associated with a DNA-damaging effect. Carvacrol and thymol reduced the level of DNA lesions induced by H(2)O(2). We assume that the observed DNA-protective effects of these plant volatiles can be associated with their antioxidative activity....

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Journal Article 2006; 27(Suppl 2): 40-43 PubMed PMID: 17159776 Citation  Keywords:  Animals, Antioxidants:analysis, Asbestos:toxicity, Cell Survival:drug effects, Chemokine CCL2:metabolism, Chemokine CCL4, Chemokines:metabolism, Glutathione Peroxidase:analysis, Glutathione Reductase:analysis, Inhalation Exposure, Lung:chemistry, Macropha.   

OBJECTIVES: Activity of cytoplasmatic superoxide dismutase, glutathione peroxidase,- and reductase, gamma-glutamyl transpeptidase and expression of macrophage chemoattractant protein-1 and macrophage inhibitory protein-1alpha was determined in primary culture of rat alveolar macrophages and type II pneumocytes after exposure to stone-wool, wollastonite and crocidolite (blue asbestos).

METHODS: The activity of redox enzymes was examined by RANDOX kits, chemokines were studied by ELISA.

RESULTS: The UICC crocidolite (positive control) decreased the activity of all redox enzymes and increased the expression of chemokines, whereas the two asbestos substituents did not alter the activity of redox enzymes either in the alveolar macrophages or pneumocytes. Stone-wool and wollastonite moderately increased the expression of chemokines in both cells.

CONCLUSIONS: The redox status and production of chemokines changed in the opposite direction, presumably owing to stronger toxic effect of asbestos. These data suggest that stone-wool and wollastonite, as potential substituents for asbestos, are less toxic than the human carcinogenic and fibrogenic crocidolite....

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Journal Article 2006; 27(Suppl 2): 35-39 PubMed PMID: 17159775 Citation  Keywords:  Animals, Coloring Agents:pharmacokinetics, Cytochrome P-450 CYP1A1:metabolism, Cytochromes b5:physiology, DNA:metabolism, DNA Adducts:metabolism, Humans, Male, Metabolic Detoxication, Phase I, Models, Biological, Naphthols:metabolism, Rabbits, Rats, Rats,.   

OBJECTIVES: Modulation of the cytochrome P450 (CYP) 1A1-mediated oxidative activation and detoxication of carcinogenic Sudan I by the heme-protein cytochrome b(5) (b(5)) was investigated. Another aim of the study was to examine the formation of Sudan I-DNA adducts in vivo.

METHODS: High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was employed for the separation of Sudan I metabolites formed by human recombinant CYPs and rat CYP1A1. The (32)P-postlabeling technique was utilized to determine Sudan I-DNA adducts.

RESULTS: The capabilities of the most efficient CYP enzymes oxidizing Sudan I, human and rat recombinant CYP1A1, as well as of human recombinant CYP1A2, 2A6 and 3A4 were significantly increased by b(5), while reactions catalyzed by human CYP1B1, 2C8, 2C9 and 2E1 were insensitive to this heme protein. Sudan I oxidation catalyzed by CYP2B6, 2C19 and 2D6 was even decreased by b(5). The stimulation of the CYP1A1-mediated Sudan I oxidation was dependent on concentration of b(5). Likewise, the increase in CYP1A1-mediated formation of Sudan I-DNA adducts by b(5) was concentration dependent. Other proteins containing heme such as cytochrome c or myoglobin were without this effect. The major Sudan I-DNA adducts formed in vitro are also generated in vivo, in livers of rats treated with Sudan I.

CONCLUSIONS: The data are the first report on the stimulation of CYP1A1-mediated oxidative reactions by b(5). In addition, the results demonstrating covalent binding of Sudan I to rat liver DNA in vivo indicate a genotoxic mechanism of Sudan I carcinogenicity in rats....

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Comparative Study 2006; 27(Suppl 2): 31-34 PubMed PMID: 17159774 Citation  Keywords:  Hyaluronic Acid:chemistry, Hydrogen Peroxide:pharmacology, Manganese Compounds:pharmacology, Models, Biological, Molecular Weight, Oxides:pharmacology, Peroxynitrous Acid:chemical synthesis, Viscosity,.   

OBJECTIVES: This contribution presents the results of the kinetics of HA degradation by peroxynitrite, which represents one of the main reactive oxygen species degrading various biomacromolecules under inflammatory conditions.

METHODS: Two simple procedures have been adapted to prepare sodium peroxynitrite: the first containing an excess of H(2)O(2), and the second in which the H(2)O(2) excess had been decomposed by MnO(2) treatment. The kinetics of hyaluronan degradation by action of peroxynitrite was monitored by rotational viscometry.

RESULTS: High-molecular-weight hyaluronan was degraded by peroxynitrite. The degradation was increased in the presence of ONOO(-) previously treated by MnO(2) in order to remove residual hydrogen peroxide. One of the reasons of this finding could be that by the action of the residual metal the pathway of ONOO(-) decomposition starts to be manifested immediately on mixing traces of metals originally present in the HA sample with the ions of manganese.

CONCLUSIONS: Trace amounts of transition metal(s) should be taken into consideration on evaluating the experimental results. Purchase of the marketed peroxynitrite product appears to be the appropriate approach to simplify and standardize the quality of ONOONa....

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Journal Article 2006; 27(Suppl 2): 27-30 PubMed PMID: 17159773 Citation  Keywords:  Adenine:analogs & derivatives, Bacteria:ultrastructure, Cell Membrane:metabolism, Cytochrome P-450 Enzyme System:genetics, Drug Interactions, Humans, Liver:drug effects, Metabolic Detoxication, Phase I, Models, Biological, NADPH-Ferrihemoprotein Reductase.   

OBJECTIVES: To determine a possible influence of acyclic nucleoside phosphonates on FMO3 and CYP activity at molecular level in vitro.

METHODS: Inhibition of individual CYP and FMO3 activities in the reconstituted system and in artificial membranes (bactosomes) was studied.

RESULTS: Activity of FMO3 was inhibited by PMPA and bisPOC-PMPA even at low levels of these drugs (below 100 microM). In reconstituted system with CYP2C9, no inhibition of CYP2C9 activity was observed. On the other hand, experiments based on membrane coexpressed system showed a modest extent of inhibition (for PMEA, PMPA, bisPOM-PMEA and bisPOC-PMPA the level of inhibition was 77.8%; 74.1%; 64.2% and 68.6%, respectively at 400 microM).

CONCLUSIONS: PMPA and bisPOC-PMPA are able to inhibit FMO3 activity at relatively low levels (10-100 microM) indicating a relatively specific interaction. Activity of CYP2C9 was affected when using the membranes coexpressed with NADPHcytochrome P450 reductase. This is probably due to more natural conditions for maintaining the CYP activity in bacterial membranes. As the inhibitor concentration in the systemic circulation does not exceed 2 microM, the probability of a significant in vivo effect of adefovir, tenofovir and the respective prodrugs on the microsomal system of cytochromes P450 and FMO3 is relatively low....

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Evaluation Studies 2006; 27(Suppl 2): 23-26 PubMed PMID: 17159772 Citation  Keywords:  Animals, Antioxidants:analysis, Body Weight:drug effects, Bronchoalveolar Lavage Fluid:chemistry, Ceramics:toxicity, Glutathione Peroxidase:analysis, Inhalation Exposure:analysis, Lung:chemistry, Lung Diseases:chemically induced, Male, Mineral Fibers:toxi.   

OBJECTIVES: The programmes of asbestos replacement brought the need to use other fibres for insulation or reinforcement of material. The aim of the presented study was to follow the effect of refractory ceramic fibres (RCF3) alone or in combination with cigarette smoke (CS) on antioxidant status of the lung in experiment on animals. As free radicals are supposed to play a role in pathogenesis of lung diseases and the toxicity of particles has been associated with production of reactive oxygen species, the antioxidant status may serve as marker of lung injury. Our hypothesis was that the effect of combined exposure to RCF3 and CS will be additive or synergic.

DESIGN: Scheme of experiment: Wistar rats were randomly divided into 4 groups per 6 animals: control, intratracheal exposure to 4 mg of RCF3, inhalatory exposure to mainstream of cigarette smoke from 8 standard research 1R1 cigarettes per day, and both intratracheal exposure to RCF3 and inhalatory to CS. The exposure lasted 6 months, the inhalatory exposure was performed 5 times per week. After finishing the exposure bronchoalveolar lavage of lungs was performed and ascorbic acid, superoxide dismutase, glutathione and glutathione peroxidase were determined in lung tissue and cell free fraction of bronchoalveolar lavage fluid (BALF).

RESULTS: The results showed that the most sensitive indicator of changes in antioxidant status was glutathione, which was changed in all groups both in BALF and lung tissue homogenate....

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Evaluation Studies 2006; 27(Suppl 2): 18-22 PubMed PMID: 17159771 Citation  Keywords:  Animals, Antineoplastic Agents:pharmacokinetics, Cytochrome P-450 Enzyme System:chemistry, DNA Adducts:metabolism, Ellipticines:pharmacokinetics, Metabolic Detoxication, Phase I, Microsomes, Liver:chemistry, Models, Biological, NADP:chemistry, NADPH-Ferri.   

OBJECTIVES: Ellipticine is a potent antineoplastic agent exhibiting multiple action mechanisms. Recently, we found that after cytochrome P450 (CYP)-mediated oxidation ellipticine forms covalent DNA adducts. Ellipticine oxidation by isolated CYP and its binding to DNA is the target of this study.

METHODS: High performance liquid chromatography (HPLC) was employed for separation and characterization of ellipticine metabolites generated by CYPs. The (32)P-postlabeling technique was utilized to determine ellipticine-DNA adducts.

RESULTS: Purified CYP enzymes reconstituted with NADPH:CYP reductase oxidized ellipticine to up to five metabolites, 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only CYP1A1 was capable to form all metabolites. Using the reconstituted enzymatic system, we demonstrated that the detoxication ellipticine metabolites, 7-hydroxyellipticine and 9-hydroxyellipticine, are mainly generated by CYP1A1 and 1A2, while those responsible for DNA binding, 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, by CYP3A1 and 2C3. Likewise, the most efficient CYPs forming DNA adducts from ellipticine were CYP3A1 and 2C3.

CONCLUSIONS: The results showed that the system of purified CYPs reconstituted with NADPH: CYP reductase proved for ellipticine oxidation provide a true reflection of the situation in the microsomal membrane....

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Journal Article 2006; 27(Suppl 2): 14-17 PubMed PMID: 17159770 Citation  Keywords:  Adenine:metabolism, Animals, Colon:drug effects, Cytochrome P-450 CYP1A1:metabolism, DNA, Single-Stranded:metabolism, Electrochemistry, Enzyme Activation:drug effects, Flavonoids:metabolism, Guanidine:metabolism, Male, Microsomes, Liver:drug effects, Mode.   

OBJECTIVES: Flavonoids are widely accepted as health promoting phytochemicals, however, some flavonoids show ability of direct interaction with DNA and/or enhance carcinogen activation into DNA modifying agents. Thus, their potential harmful effect on the human body should be examined in detail.

METHODS: Direct interaction of flavonoids (quercetin, rutin) with DNA was examined using square wave voltammetry on carbon paste electrode. Induction effect of selected flavonoids on content of cytochrome P450 1A1, carcinogens activating enzyme, in colon and liver microsomal samples of animals exposed to flavonoids was determined by Western blotting, using anti-cytochrome P450 1A1 specific antibody.

RESULTS: Of the natural flavonoids tested, induction of CYP1A1 was elicited by the typical citrus flavonoid naringenin in the colon, as well as by flavone in the liver. Moreover, synthetic beta-naphthoflavone and naturally occurring chrysin, quercetin and diosmin induced CYP1A1 in both tissues. The oxidation signals of guanine and adenine in the DNA molecule were decreased in the presence of flavonoids.

CONCLUSIONS: Although flavonoids are often considered to be safe because of their "plant origin", ingestion of flavonoids should be taken with caution. Enhanced expression of CYP1A1 in colon tissue might be responsible for increasing incidence of colorectal carcinoma in humans. Electrochemistry can be used to study the interactions of flavonoids and DNA....

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