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Journal Article 2014; 35(Suppl 2): 123-132 PubMed PMID: 25638376 Citation  Keywords:  Animals, Aristolochic Acids:metabolism, Dicumarol:pharmacology, Enzyme Inhibitors:pharmacology, Humans, Quinone Reductases:metabolism, Rats,.   

ong>OBJECTIVES:ong> Dicoumarol is known to act as an inhibitor of

ong>NAD(P)H:ong> quinone oxidoreductase (NQO1). This cytosolic reductase significantly contributes to the genotoxicity of the nephrotoxic and carcinogenic alkaloid aristolochic acid I (AAI). Aristolochic acid causes aristolochic acid nephropathy (AAN), and Balkan endemic nephropathy (BEN), as well as associated urothelial malignancies. NQO1 is the most efficient enzyme responsible for the reductive bioactivation of AAI to species forming covalent AAI-DNA adducts. However, it is still not known how dicoumarol influences the NQO1-mediated reductive bioactivation of AAI.

ong>METHODS:ong> AAI-DNA adduct formation was determined by 32P-postlabeling. Expression of NQO1 mRNA and NQO1 protein was determined by real-time polymerase chain reaction and Western blotting, respectively.

ong>RESULTS:ong> In this study, dicoumarol inhibited AAI bioactivation to form AAI-DNA adducts mediated by rat and human NQO1 in vitro as expected. We however, demonstrated that dicoumarol acts as an inducer of NQO1 in kidney and lung of rats treated with this NQO1 inhibitor in vivo, both at protein and activity levels. This NQO1 induction increased the potency of kidney cytosol to bioactivate AAI and elevated AAI-DNA adduct levels were found in ex-vivo incubations of AAI with renal cytosols and DNA. NQO1 mRNA levels were induced in liver only by dicoumarol.

ong>CONCLUSION:ong> Our results indicate a dual role of dicoumarol in NQO1-mediated genotoxicty of AAI. It acts both as an NQO1 inhibitor mainly in vitro and as an NQO1 inducer if administered to rats.

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Journal Article 2014; 35(Suppl 2): 114-122 PubMed PMID: 25638375 Citation  Keywords:  Animals, Aryl Hydrocarbon Hydroxylases:metabolism, Cell Membrane:metabolism, Cytochrome P450 Family 2, Cytochromes b5:metabolism,.   

ong>OBJECTIVES:ong> The mammalian mixed function oxidase (MFO) system participates in hydroxylation of many hydrophobic endogenous compounds as well as xenobiotics such as drugs and carcinogens. This biotransformation system, located in a membrane of endoplasmic reticulum, consists of cytochrome P-450 (P450), NADPH:P450 oxidoreductase and a facultative component, cytochrome b5. The knowledge of the interactions among the individual components of the MFO system is essential to understand the relationships between the structure and function of this system that finally dictate a qualitative and quantitative pattern of produced metabolites (e.g. detoxified xenobiotics and/or activated carcinogens). To elucidate the quantitative aspects of the interactions within the MFO system we acquired the photo-initiated cross-linking approach.

ong>METHODS:ong> The photo-initiated cross-linking employing cytochrome b5 as a protein nanoprobe [an amino acid analogue of methionine (pMet) was incorporated into cytochrome b5 sequence during recombinant expression] was used to quantify its interaction with P450 2B4 in a functional membrane complex. The cross-linking was initiated by UV-irradiation that formed from a pMet photolabile diazirine group highly reactive carbene biradical. This biradical is able to covalently bind amino acids in the close proximity and to form cross-link. The Met 96 of cytochrome b5 is situated in a linker region between its catalytic and membrane domains, while Met 126 and 131 are located in its membrane domain. The combination of several methods (electrophoresis in polyacrylamide gel, isoelectric focusing, Edman N-terminal degradation and amino acid analysis) was employed to characterize the molar ratio of P450 2B4 to cytochrome b5 in formed covalent cross-links to quantify their transient interactions.

ong>RESULTS:ong> The successfully produced cytochrome b5 nanoprobe (with confirmed pMet incorporation by mass spectrometry) stimulates the catalytical activity of P450 2B4 when reconstituted with NADPH:P450 oxidoreductase in vitro in dilauroylphosphatidylcholine (DLPC) vesicles. The cross-linking was carried out in similar reconstituted system without NADPH:P450 oxidoreductase, and at least three products were separated on 1D SDS-PAGE. The molar ratio of P450 to cytochrome b5 in each complex was estimated using the above-mentioned combination of methods as 1:1, 1:2 and 2:1.

ong>CONCLUSIONS:ong> The results demonstrate the utility of cytochrome b5 nanoprobe to study the interactions in MFO system. Using this nanoprobe, heterodimer with P450 2B4 and in addition also heterooligomers were identified, suggesting rather complex interactions of both proteins in this system that suppose the formation of such multimeric structures in the membrane of endoplasmic reticulum.

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Journal Article 2014; 35(Suppl 2): 105-113 PubMed PMID: 25638374 Citation  Keywords:  Benzo(a)pyrene:metabolism, Cytochrome P-450 CYP1A1:metabolism, Cytochromes b5:metabolism, Humans, NADH, NADPH Oxidoreductases:metabolism,.   

ong>OBJECTIVES:ong> Cytochrome P450 (CYP) 1A1 located in the membrane of endoplasmic reticulum is the most important enzyme in both activation and detoxification of carcinogenic benzo[a]pyrene (BaP), in combination with microsomal epoxide hydrolase (mEH). However, it is still not clearly explained how the electron transfer is mediated by NADPH:CYP oxidoreductase (POR), another component of the microsomal enzymatic system, on CYP1A1 during BaP oxidation, and whether microsomal cytochrome b5 might influence this electron transfer.

ong>METHODS:ong> High performance liquid chromatography (HPLC) was employed for separation of BaP metabolites formed by enzymatic systems containing human CYP1A1.

ong>RESULTS:ong> Human CYP1A1 expressed with POR in eukaryotic and prokaryotic expression cellular systems, in microsomes of insect cells (Supersomes) and in a membrane fraction of Escherichia coli, respectively, and these enzyme systems reconstituted with purified cytochrome b5 were utilized to study BaP oxidation. Human CYP1A1 expressed in Supersomes oxidized BaP to seven metabolites [7,8- and 9,10-dihydrodiols, 1,6-dione, 3,6-dione, 3- and 9-phenols, and a metabolite with unknown structure (Mx)], whereas this enzyme expressed in membranes of E. coli formed only the metabolites 1,6- and 3,6-diones, 3- and 9-phenols, and Mx. Addition of cytochrome b5 to CYP1A1 expressed in the eukaryotic system led to a more than 2-fold increase in BaP metabolism, but had essentially no effect on BaP oxidation by CYP1A1 expressed in E. coli.

ong>CONCLUSION:ong> The effect of cytochrome b5 on CYP1A1 conformation and the electron transfer to this enzyme may contribute to the cytochrome b5-mediated stimulation of BaP oxidation.

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Journal Article 2014; 35(Suppl 2): 99-104 PubMed PMID: 25638373 Citation  Keywords:  Animals, Antibodies:immunology, Bacterial Infections:prevention & control, Cell Line, Chickens, Egg Yolk:immunology, Epithelial Cells:immunology, Humans, Immunoglobulin Fab Fragments:immunology, Immunoglobulins:immunology, Inflammation:immunology, Lung:im.   

ong>OBJECTIVES:ong> As an alternative therapeutic approach for the prevention and treatment of bacterial infections with P. aeruginosa of cystic fibrosis (CF) patients, chicken yolk antibodies (IgY) could be used. The most significant advantage of IgY, in contrast to mammalian IgG, consists in the fact, that when bound to the antigen, they usually do not induce inflammatory reaction. In addition, the simplicity of egg production and the ease of IgY preparation makes this kind of antibody an excellent tool for passive immunization. Thus, the aim of our project was to study the effect of IgY and its Fab fragment on the potential induction of pro-inflammatory reactions in lung epithelial cells.

ong>METHODS:ong> Chicken IgY were prepared from pooled egg yolks. Fab fragmens of IgY were purified from the papain digest of IgY using DEAE-Sephacel ion exchange chromatography. Their purity was verified by SDS electrophoresis in polyacrylamide gel. Immortalized human cell lines, CuFi (CF patient) and NuLi (healthy subject), and A549 (human adenocarcinoma cells) were exposed to IgY, Fab, OVA, LPS (positive control), PBS (negative control), and human and goat IgG for 24 hours. The concentration of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and GM-CSF were determined in cell media using the BioPlex method, which enables the quantification of multiple analytes simultaneously in one sample.

ong>RESULTS:ong> Our results show that i) the Fab fragment induced levels of some proinflammatory cytokines, when compared to the PBS control, whereas ii) chicken IgYs did not induce any notable production of pro-inflammatory cytokines in contrast to intense effect of LPS on TNF-α and GM-CSF. In summary, our data show that levels of all cytokines are comparable with physiological values in human serum except of IL-1β, which concentration in cell medium was markedly elevated by Fab fragment.

ong>CONCLUSIONS:ong> The present data indicate that IgY are not inflammatory for lung cells and thus they are possibly applicable for prevention of airway bacterial infections.

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Journal Article 2014; 35(Suppl 2): 93-98 PubMed PMID: 25638372 Citation  Keywords:  Animals, Astacoidea:drug effects, Embryo, Nonmammalian:drug effects, Herbicides:toxicity, Prometryne:toxicity,.   

ong>OBJECTIVES:ong> The aim of this study is to assess the toxicity of prometryne in early life stages of marbled crayfish (Procambarus fallax f. virginalis) on the basis of mortality, early ontogeny, growth rate, and histopathology during and at the end of the test.

ong>DESIGN:ong> The early life stages of marbled crayfish were exposed to prometryne at four concentrations, 0.51, (reported concentration in Czech rivers), 144, 1440, and 4320 μg x l(-1) for 53 days and compared to crayfish in a non-treated control group. RESU LTS: Prometryne in concentration 144, 1444 and 4320 μg x l(-1) caused decrease of weight and specific growth rates of crayfish. Crayfish exposed the highest concentration 4320 μg x l(-1) showed delay in ontogeny development. All crayfish groups exposed to prometryne showed histopathological changes in gill. On the basis of histopathological changes the values of LOEC = 0.51 μg x l(-1) and NOEC = for 0.10 μg x l(-1) of prometryne for marbled crayfish juveniles was estimated.

ong>CONCLUSIONS:ong> Chronic exposure of prometryne on early life stages of crayfish has affected their mortality, growth rate, and histology. Some of the changes were observed only at higher exposures (144, 1444 and 4320 μg x l(-1)), but histopathological changes in gills were observed also in crayfish exposed to the real environmental concentration in Czech rivers (i.e. 0.51 μg x l(-1)), which is about 9 times lower than maximal concentration (4.40 μg x l(-1)) reported in surface waters of Greece. Concentrations of prometryne in World rivers have been reported to generally vary in the range of 0.1-4.40 μg x l(-1).

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Journal Article 2014; 35(Suppl 2): 86-92 PubMed PMID: 25638371 Citation  Keywords:  Animals, Carps:blood, Oxidants:pharmacology, Peracetic Acid:pharmacology,.   

ong>OBJECTIVES:ong> The aim of present study was to evaluate the effect of peracetic acid (PAA) on haematotological and biochemical indices, antioxidant status, micronucleus induction and histopathological alterations of liver and gill in grass carp.

ong>METHODS:ong> Grass carp (Ctenopharyngodon idella) juveniles were exposed to therapeutic concentrations (1, and 3 mg x l(-1)) of PAA for a period of 10 days. Selected haematotological indices--the erythrocyte count (RBC), haematocrit (PCV), haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and leukocyte count (WBC), and biochemical indices--glucose (Glu), total protein (TP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), and lactate dehydrogenase (LDH) were evaluated in plasma. Activity of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), as well as levels of thiobarbituric acid reactive substances (TBARS) were assessed in gill and liver. Micronucleus frequency in peripheral erythrocytes was counted in control and experimental fish. Histological examinations of gill and liver were performed.

ong>RESULTS:ong> No significant differences were found in haematological parameters measured. Statistically significant (p < 0.05) alterations in the activities of AST, CK and LDH were found in treated fish compared to control groups. Fish exposed to 1 mg x l(-1) of PAA showed significantly lower (p < 0.05) SOD activity in liver and gill while catalase activity indicated a significant decrease (p < 0.05) only in gill tissue. Other significant changes were observed in GR activity in gill in both PAA exposed groups, while GR activity in liver remained unchanged. There was no significant difference in the count of micronuclei between control and exposed fish. Haemorrhage, fusion of primary lamellae, degeneration of secondary lamellae, some clubbing on primary and secondary lamellae tips, and lifting of epithelial cells were found in gill tissues in both control and treated fish.

ong>CONCLUSION:ong> The results show that PAA could induce alterations in biochemical parameters in blood plasma, antioxidant enzymes response and histopathological changes in gill; however, it seems that these changes are reversible. Subsequently, lower concentration (1 mg x l(-1)) is useable as a treatment concentration for grass carp.

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Journal Article 2014; 35(Suppl 2): 81-85 PubMed PMID: 25638370 Citation  Keywords:  Aminobenzoates:pharmacology, Anesthetics:pharmacology, Animals, Clove Oil:pharmacology, Dose-Response Relationship, Drug, Etomidate:pharmacology, Perches, Temperature, Water,.   

ong>OBJECTIVES:ong> The objectives of the study were to compare the different doses of clove oil, Propiscin, and tricaine methane sulphonate (MS 222) in relation to water temperature in pikeperch aquaculture.

ong>DESIGN:ong> For assessment of this experiment 168 fish (10.77 ± 0.59 cm total body length and 7.88 ± 1.74 g body weight) were used. Three different anaesthetic treatments (Propiscin, clove oil and MS 222) were used. Three doses of each anaesthetic treatment (Propiscin: 0.5; 1; 1.5 ml x L(-1), clove oil: 15; 30; 60 mg x L(-1), MS 222: 50; 100; 150 mg x L(-1)) were compared at three different temperatures 9.5; 15.5 and 23 degrees C.

ong>RESULTS:ong> In comparison of these doses of anaesthetic in different temperature, the significantly shortest time to attain phase A7 (total complete anaesthesia) was observed for Propiscin (1.5 ml L(-1)) 0:31 ± 0:04 min (23 degrees C) to 0:33 ± 0:25 min (9.5 degrees C) compared to MS 222 (150 mg x L(-1)) 1:04 ± 0:21 min (23 degrees C) to 1:54 ± 0:32 min (9.5 degrees C) and clove oil (60 mg x L(-1)) 1:05 ± 0:17 min (23 degrees C) to 3:05 ± 0:31 min (9.5 degrees C). On the other hand, the longest time of anaesthesia recovery was attained using Propiscin (1.5 ml x L(-1)) 10:35 ± 1:40 min (23 degrees C) to 32:30 ± 1:10 min (9.5 degrees C) compared to clove oil (60 mg x L(-1)) 2:39 ± 0:50 min (23 degrees C) to 9:36 ± 2:34 min (60 mg x L(-1), 9.5 degrees C) and MS 222 (150 mg x L(-1)) 2:26 ± 1:27 min (23 degrees C) to 4:59 ± 0:39 min (9.5 degrees C).

ong>CONCLUSIONS:ong> The results from this study showed that the optimal and sufficient doses in all tested temperatures for pikeperch are 30 mg x L(-1) of clove oil, 100 mg x L(-1) of MS 222 and 0.5 ml x L(-1) of Propiscin.

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Journal Article 2014; 35(Suppl 2): 71-80 PubMed PMID: 25638369 Citation  Keywords:  Animals, Antioxidants:pharmacology, Carotenoids:pharmacology, Carps:metabolism, Chloramines:pharmacology, Diet, Oxidative Stress:drug effects, Sulfonamides:pharmacology, Tosyl Compounds:pharmacology,.   

ong>OBJECTIVES:ong> In fish aquaculture, disinfectants are used against bacterial and protozoal infections. These compounds cause oxidative stress that may stimulate the generation of reactive oxygen species, and subsequently the alteration in antioxidant systems of exposed organisms. Antioxidants like carotenoids present in microalgae increase carp resistance to oxidative stress after chemical treatment.

ong>DESIGN:ong> The aim of these experiments was to prove increased resistance of common carp (Cyprinus carpio L.) juveniles fed on experimental diets with microalgae biomass supplement (Algadiets) to oxidative stress caused by a disinfectant chloramine-T. In indoor experiments fish were fed on laboratory-prepared extruded diets containing supplement of Chlorella spp. (cf. C. vulgaris Beijerinck) biomass which contains antioxidants (carotenoids) like lutein. The young-of-the-year-old fish were acclimatized and fed on basal diet (control group) and the on diets containing 1, 2, 5 and 10% (w/w) of spray-dried Chlorella biomass (Algadiet 1, 2, 5 and 10) for 14 days followed by 6 weeks. Consequently, fish were treated daily with chloramine-T (Chl-T) at concentration of 10 mg x l(-1) for 1 h in three consecutive days. After this treatment, the indices of oxidative stress and antioxidant enzyme activity were assayed in fish gill, muscle and hepatopancreas.

ong>RESULTS:ong> The fish fed on different Algadiets had increased antioxidant enzyme activities of glutathione peroxidase, glutathione reductase and catalase in flesh after the exposure to Chl-T. Higher activities of superoxide dismutase, glutathione peroxidase and glutathione reductase were also observed in the hepatopancreas in all tested concentrations compared to the control group fed on the basal diet. The increased production and activity of antioxidant enzymes confirmed improved protection ability of fish tissues against oxidative damage when microalgae biomass was supplemented to the fish diet which was more pronounced by higher microalgae supplement in Algadiet 5 and 10 where the content of carotenoids was 105 mg and 214 mg per kilogram of fed, respectively.

ong>CONCLUSION:ong> The results show the positive effect of carotenoids from microalgae biomass to maintain the antioxidant capacity which increases resistance of fish to oxidative stress.

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