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Editorial 2010; 31(Suppl 2): 1-3 PubMed PMID: 21187834 Citation  Keywords:  Humans, Slovakia, Toxicology:methods,.   

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Journal Article 2010; 31(Suppl 2): 46-50 PubMed PMID: 21187832 Citation  Keywords:  Animals, Aryl Hydrocarbon Hydroxylases:metabolism, Colon:enzymology, Cytochrome P-450 CYP3A:metabolism, Cytochrome P-450 Enzyme System:metabolism, Duodenum:enzymology, Escherichia coli, Gastrointestinal Tract:enzymology, Male, Models, Animal, Probiotics:p.   

OBJECTIVES: The aim of the study was to find whether probiotic Escherichia coli Nissle 1917 O6:K5:H1 (EcN) influences the expression of cytochromes P450 (CYP) in the rat intestine.

DESIGN: Live bacterial suspension of EcN was administered to healthy male Wistar rats daily for 7 days. Control group of rats was stressed by oral application of the saline solution daily for 7 days as well. Sections of the duodenum, jejunum, ileum, caecum and colon have been taken from each experimental animal. With all individual samples, microsomal fraction has been prepared and expression of selected CYPs was determined by Western blotting. The levels of expression of CYPs were also evaluated by mRNA using real-time PCR.

RESULTS: It was found that there are changes in expression of CYP enzymes studied along the intestine. CYP1A1, 2B1/2 and 2E1 are present mainly in the duodenum and jejunum; on the other hand, CYP2C6 is expressed mainly in the caecum and colon. CYP3A was found all over the rat intestine. The results show that there are no prominent differences between control samples and samples with EcN, only the expression of CYP3A protein in the duodenum appears to exhibit a clear tendency to decrease. In the case of the colon, a significant increase in the expression of CYP3A (most likely CYP3A1) after treatment of rats with EcN was found.

CONCLUSION: This in vivo study revealed that the levels of colon CYP3A could be significantly increased in rats treated with probiotic EcN. On the contrary, the expression of CYP3A in the duodenum decreased. However, the changes in the expression of CYP enzymes are probably not as extensive to be clinically important in man; hence, most likely the probiotic EcN has little influence on the intestinal drug metabolism by CYP enzymes....

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Evaluation Studies 2010; 31(Suppl 2): 16-20 PubMed PMID: 21187829 Citation  Keywords:  Animals, Dose-Response Relationship, Drug, Genes, Bacterial:drug effects, Metabolism:drug effects, Microcystins:pharmacology, Mutagenicity Tests, Neurotoxins:pharmacology, Salmonella typhimurium:genetics, Tropanes:pharmacology,.   

OBJECTIVE: The aim of this study was to evaluate genotoxicity of anatoxin-a, cyanotoxin of neurotoxic activity. Additionally, other frequently detected cyanotoxin of previously described genotoxic potential, microcystin-LR, was used at the same concentrations, as well as the mixture of both toxins, anatoxin-a and microcystin-LR.

DESIGN: Genotoxicity of the toxins was determined with the use of the umuC assay, in which the induction and expression of the umuC - lacZ reporter gene was assessed. The test was conducted on Salmonella typhimurium TA 1535/pSK1002 strain, with and without metabolic transformation. The toxin concentrations were 0.25, 0.5, 1 and 2 µg/ml. The exposure time was 2 h.

RESULTS: The highest inefficient concentration of anatoxin-a without metabolic transformation was 0.25 µg/ml, of microcystin-LR was 0.5 µg/ml and in case of the toxin mixture all used concentrations induced the umuC gene. When S9 fraction was added to the samples, no effects were detected.

CONCLUSION: To our knowledge, this is the first report on genotoxic effects of anatoxin-a. Although the study is preliminary and needs further research, however, indicates the new potential activity of the toxin, as well as the possible increase of genotoxicity of other cyanotoxins, more stable in the environment, e.g. microcystin-LR....

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Journal Article 2010; 31(Suppl 2): 79-83 PubMed PMID: 21187828 Citation  Keywords:  Animals, Antioxidants:pharmacology, Antirheumatic Agents:therapeutic use, Arthritis, Experimental:drug therapy, Cell Line, Disease Models, Animal, Drug Therapy, Combination, Macrophages:cytology, Male, Methotrexate:therapeutic use, Neutrophils:drug effect.   

OBJECTIVE: Formation of reactive oxygen species in neutrophils of rats with adjuvant arthritis and generation of nitric oxide in RAW 264.7 macrophages were analysed in the presence of pinosylvin.

METHODS AND RESULTS: The method of chemiluminescence was used for the detection of reactive oxygen species in blood of rats with adjuvant arthritis. Pinosylvin (50 mg/kg, daily, p.o.) and methotrexate (0.4 mg/kg, twice a week, p.o.) were applied separately or in a combination over a period of 28 days from the day of immunisation. Adjuvant arthritis was accompanied by a significantly increased number of neutrophils, by elevated concentration of oxidants in blood and by excessive responsiveness of neutrophils to stimulation with PMA. In rats treated with methotrexate, all these changes were significantly reduced and the inhibition became more pronounced when methotrexate was applied in the combination with pinosylvin; the monotherapy with pinosylvin did not induce any detectable changes in the parameters tested. Under in vitro conditions, pinosylvin inhibited formation of nitric oxide (NO) in macrophages, as demonstrated by the decreased concentration of nitrite - the end-product of NO metabolism (assessed by Griess' method), by the reduced expression of inducible NO synthase (detected by Western blot), and by the failure of pinosylvin to scavenge nitric oxide (measured amperometrically in cell-free system).

CONCLUSION: The observed ability of pinosylvin to decrease concentration of reactive oxygen and nitrogen species, along with its capacity to enhance the efficacy of methotrexate in arthritis treatment may shed more light into the pharmacological potential of this prospective natural substance....

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Journal Article 2010; 31(Suppl 2): 36-45 PubMed PMID: 21187827 Citation  Keywords:  Aniline Compounds:metabolism, Animals, Anisoles:metabolism, Aryl Hydrocarbon Hydroxylases:physiology, Carcinogens:metabolism, Cytochrome P-450 CYP2E1:physiology, Cytochrome P-450 Enzyme System:physiology, Environmental Pollutants:metabolism, Hydroxylamine.   

OBJECTIVES: N-(2-methoxyphenyl)hydroxylamine is a human metabolite of two industrial and environmental pollutants and bladder carcinogens 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole). Metabolism of N-(2-methoxyphenyl)hydroxylamine by rat hepatic microsomes and identification of the major microsomal enzymes participating in this process are aims of this study.

METHODS: HPLC with UV detection was employed for the separation of N-(2-methoxyphenyl)hydroxylamine metabolites. Inducers and inhibitors of microsomal enzymes and rat recombinant CYPs were used to characterize the enzymes participating in N-(2-methoxyphenyl)hydroxylamine metabolism.

RESULTS: N-(2-methoxyphenyl)hydroxylamine is metabolized by rat hepatic microsomes predominantly to o-anisidine, the parent carcinogen from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome b5 reductase and hepatic microsomes of rats pre-treated with specific inducers of CYPs and NADPH:CYP reductase were used to characterize rat liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute most of N-(2-methoxyphenyl)hydroxylamine metabolism to o-anisidine in rat liver to CYP2C, followed by CYP2E1, 2D and 2A. Among recombinant rat CYP enzymes tested in this study, rat CYP2C11 and 2E1, followed by CYP2A2, 2D1/2, 2C12, 3A1/2 and 1A1/2 were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine.

CONCLUSION: The results found in this study, the first report on the reduction of N-(2-methoxyphenyl)hydroxylamine by rat CYP enzymes, demonstrate that CYP2C, followed by CYP2E1, 2D and 2A are the major enzymes participating in this process in rat liver....

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Journal Article 2010; 31(Suppl 2): 91-95 PubMed PMID: 21187826 Citation  Keywords:  Animals, Anti-Inflammatory Agents:pharmacology, Antioxidants:pharmacology, Arthritis, Experimental:drug therapy, Disease Models, Animal, Dose-Response Relationship, Drug, Drug Therapy, Combination, Male, Oxidative Stress:drug effects, Peroxidase:metabolis.   

OBJECTIVE: The aim of this study was to evaluate the effects of pinosylvin (PIN) and pterostilbene (PTE), natural substances from the stilbenoid group, on the development of adjuvant arthritis in rats.

METHODS: Adjuvant arthritis (AA) was induced by a single intradermal injection of Mycobacterium butyricum in incomplete Freund's adjuvant in male Lewis rats. Our experiments included healthy intact animals as reference controls, arthritic animals without any drug administration, and arthritic animals with administration of PIN and PTE in the oral daily dose of 30 mg/kg b.w. The treatment involved administration of the substances tested from day 0, i.e. the day of immunization, to the experimental day 28. The following parameters were monitored: change of the hind paw volume (HPV) on day 14, 21 and 28, luminol-enhanced chemiluminescence (CL) of the joint and myeloperoxidase (MPO) activity in hind paw joint homogenates (day 28).

RESULTS: Arthritic animals treated with PIN showed a decrease in HPV, significantly on days 14 and 28. PIN decreased CL of the joint as well as MPO activity of the joint homogenate, in comparison with untreated animals. PTE had no effect on HPV and MPO activity in hind paw joint homogenates and exerted only a partial effect on luminol-enhanced CL.

CONCLUSIONS: On the basis of our results we conclude that the effect of PTE on CL was only partial. PIN, on the other hand, had a beneficial anti-inflammatory and antioxidant effect on oxidative stress induced biochemical changes occurring in AA, as determined by all three functional parameters....

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Evaluation Studies 2010; 31(Suppl 2): 62-68 PubMed PMID: 21187825 Citation  Keywords:  Animals, Antidotes:pharmacology, Antioxidants:pharmacology, Glutathione Reductase:metabolism, Liver:drug effects, Male, Models, Animal, Oxidative Stress:drug effects, Oximes:pharmacology, Pyridinium Compounds:pharmacology, Radiation, Ionizing, Rats, Rats,.   

OBJECTIVES: HI-6 is an antidotum suitable for treatment of intoxication by nerve agents. The recent investigation appointed its modulation of inflammatory response as well as vegetative nervous system activity. However, the present experiments were carried out in order to assess the antioxidant effect of HI-6 in irradiated animals.

METHODS: male Wistar rats were irradiated by ionizing radiation (7.5 Gy, LD50/30). Animals were divided into four groups: i.e. controls (A), irradiated (B), treated with HI-6 (C), and both irradiated and treated with HI-6 (D). Ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS) and glutathione reductase activity were assayed in liver, spleen, plasma, and whole blood. Clinical biochemistry markers were determined in plasma samples.

RESULTS: We found significantly increased FRAP levels in liver, while its levels decreased in the spleen of B group animals. Ionising radiation (B group) also significantly elevated TBARS values in spleen. HI-6 reversed FRAP and TBARS values to control levels. Glutathione reductase activity was significantly elevated in spleen and liver of animals exposed to HI-6 (C and D groups). Clinical biochemistry markers were shifted only slightly. The in vitro test confirmed the inhibitory effect of HI-6 towards acetylcholinesterase.

CONCLUSIONS: In conclusion, HI-6 is potent in suppressing oxidative stress and might be a promising drug in the field of radiation protection....

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Journal Article 2010; 31(Suppl 2): 84-90 PubMed PMID: 21187824 Citation  Keywords:  Antioxidants:pharmacology, Apoptosis:drug effects, Caspase 3:metabolism, Cell Survival:drug effects, Computer Simulation, Dose-Response Relationship, Drug, Humans, In Vitro Techniques, Male, Neutrophils:cytology, Phosphorylation:drug effects, Protein Kina.   

OBJECTIVE: Pterostilbene, a naturally occurring phenolic derivative, exhibits various pharmacological effects, e.g. anti-cancerous, antioxidant, anti-inflammatory and anti-diabetic. Based on our previous study, we assessed the cellular and molecular effects of pterostilbene on human neutrophils and in cell free systems. Experimental and theoretical molecular descriptors of stilbene derivatives were also determined.

METHODS: We assessed the antioxidant properties of pterostilbene using cell free system and computational methods. The effect of pterostilbene on protein kinase C activation/phosphorylation was detected by special anti-phospho protein kinase C antibodies. Membrane associated changes determining the life span of neutrophils and human recombinant caspase-3 assay were examined.

RESULTS: Pterostilbene possessed comparable antioxidant properties as resveratrol in cell free system. Computational methods were used to establish the molecular characteristics of stilbene derivatives. The values of electronic parameters suggest a slight enhancement of electron donor properties of pterostilbene compared to resveratrol. Phosphorylation and thus activation of protein kinase C alpha/beta II in activated neutrophils was not decreased by pterostilbene. Pterostilbene in concentrations of 10-100 μM was found to inhibit the activity of human caspase-3 purified enzyme and did not influence cell viability significantly.

CONCLUSION: Pterostilbene, an analog of resveratrol, was identified as a good natural antioxidant compound. However, reducing the oxidative burst of human neutrophils during their activation in vitro with pterostilbene does not include protein kinase C phosphorylation pathway. Pterostilbene showed dose dependent activation/inhibition of caspase-3 enzyme activity....

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