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Comparative Study 2012; 33(Suppl 3): 66-71 PubMed PMID: 23353846 Citation  Keywords:  Aminobenzoates:toxicity, Anesthetics:pharmacology, Animals, Aquaculture:methods, Blood Proteins:metabolism, Clove Oil:pharmacology, Erythrocyte Count, Esocidae:blood, Ethylene Glycols:pharmacology, Etomidate:pharmacology, Hematocrit, Perches:blood,.   

OBJECTIVES: The objectives of the study were to compare the effects of Propiscin, 2-phenoxyethanol, clove oil and tricaine methane sulphonate (MS 222), anaesthetics frequently used in aquaculture.

DESIGN: The haematological and biochemical blood profiles of pikeperch (Sander lucioperca L.) anesthetized with Propiscin (1.5 ml L-1), 2-phenoxyethanol (0.3 ml L-1), clove oil (33 mg L-1), MS 222 (150 mg L-1) and non-anesthetized control group were tested. Each tested group was divided into two subgroups, the first subgroup was sampled in anaesthesia 10 min after application of the anaesthetic and the second one live on 24h.

RESULTS: The erythrocyte count and haematocrit was significantly decreased in 2-phenoxyethanol (24 h) compared with control group (CG). The mean corpuscular haemoglobin concentration was significantly increased in 2-phenoxyethanol (10 min), Propiscin (10 min and 24 h) compared to CG. The 2-phenoxyethanol (10 min and 24 h), MS 222 (24 h), clove oil (24 h), and Propiscin (10 min and 24 h) showed significantly lower leukocyte count compared with CG. The level of glucose was significantly (p<0.05) elevated with MS 222 (10 min) and clove oil (10 min) compared with CG. The 2-phenoxyethanol (10 min and 24 h), MS 222 (24 h), clove oil (24 h), and Propiscin (24 h) showed significantly lower (p<0.01) ammonia levels compared with CG. The triacylglycerols was significantly decreased (p<0.01) with Propiscin (10 min and 24 h), MS 222 (24 h), clove oil (24 h) and with 2-phenoxyethanol (24 h) compared with CG. After 24 hours MS 222 (24 h) and Propiscin (24 h) anaesthesia, fish showed significantly lower (p<0.01) concentration of inorganic phosphate compared with CG.

CONCLUSIONS: On the basis of this experiment, it appears that clove oil was associated with the lowest effects in pikeperch and therefore would be recommended as an alternative to MS 222, while Propiscin and 2-phenoxyethanol are not suitable for manipulation with pikeperch in aquaculture....

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Comparative Study 2012; 33(Suppl 3): 60-65 PubMed PMID: 23353845 Citation  Keywords:  Abnormalities, Multiple:chemically induced, Acetone:toxicity, Animals, Dimethyl Sulfoxide:toxicity, Dose-Response Relationship, Drug, Edema:chemically induced, Embryo, Nonmammalian:drug effects, Ethanol:toxicity, Eye Abnormalities:chemically induced, Meth.   

OBJECTIVES: The aim of this study was to determine the effects of 96 hour exposure to selected solvents on the embryonic stages of zebrafish (Danio rerio). We investigated mortality and various types of changes which appeared (oedema, tail and eye defects, weak pigmentation, and deformation of the body). Based on the results, values of NOEC and LOEC for embryos of D. rerio were determined.

METHODS: Embryonal toxicity tests were conducted according to OECD guideline 212. Ethanol and methanol were tested at concentrations of 0.1, 0.5, 1, 1.5, 2%; acetone at concentrations of 0.1, 0.5, 1, 1.25, 1.5%; and dimethylsulfoxide at concentrations of 1, 1.5, 2, 2.5, 3%.

RESULTS: The LOEC values of ethanol and methanol were detected in the 1% concentration. Statistically significant changes (oedema) were reported in 1% ethanol, and oedema, weak pigmentation and deformation of the body were observed in 1% methanol. After exposure to acetone, the most common occurrence of oedema was in the 0.5% concentration (LOEC = 0.5%). The solvent dimethylsulfoxide caused oedema and body deformation at the 2% concentration (LOEC = 2%).

CONCLUSIONS: The NOEC concentrations of the individual solvents were as follows: ethanol and methanol, 0.5%; acetone, 0.1%; and dimethylsulfoxide, 1.5%. These concentrations of individual solvents were higher than the maximum recommended concentration for toxicity tests on fish. For this reason, it can be assumed that the concentration of solvent allowed by the norm does not affect the procedure or results of such tests....

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Journal Article 2012; 33(Suppl 3): 48-52 PubMed PMID: 23353843 Citation  Keywords:  Animals, Aryl Hydrocarbon Hydroxylases:antagonists & inhibitors, Cholesterol, Dietary:pharmacology, Cytochrome P-450 CYP2C9, Cytochrome P450 Family 2, Fluorobenzenes:pharmacology, Gene Expression Regulation, Enzymologic:drug effects, Humans, Hydroxymethyl.   

OBJECTIVES: The aim of this study was to investigate whether rosuvastatin affects expression and activity of rat CYP2C6. This cytochrome P450 is considered to be a counterpart of human CYP2C9, which metabolizes many drugs, including diclofenac, ibuprofen or warfarin.

DESIGN: Male hereditary hypertriglyceridemic (HHTg) rats were fed standard laboratory diet (STD) or high cholesterol diet (HCD: STD + 1% of cholesterol w/w + 10% of lard fat w/w) for 21 days. A third group of rats were fed high a cholesterol diet with rosuvastatin added (0.03% w/w). Expression of CYP2C6 was measured in liver samples using real-time PCR (mRNA level) and Western blotting (protein level). Formation of diclofenac metabolites (typical enzyme activity of CYP2C6) was analyzed using HPLC with UV detection.

RESULTS: Administration of rosuvastatin to HHTg rats resulted in significantly increased mRNA expression and enzyme activity in HCD-fed animals; changes of CYP2C6 protein were non-significant. These results suggest that CYP2C6 expression and activity are positively affected by rosuvastatin in hereditary hypertriglyceridemic rats after intake of HCD.

CONCLUSION: The results presented open the possibility that in humans, rosuvastatin may affect the metabolism of many drugs by influencing expression and activity of CYP2C6 (counterpart of human CYP2C9). Further studies are needed to elucidate the effects of this statin on CYP2C9 in humans....

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Journal Article 2012; 33(Suppl 3): 41-47 PubMed PMID: 23353842 Citation  Keywords:  Animals, Aryl Hydrocarbon Hydroxylases:chemistry, Carbodiimides:chemistry, Chromatography, Liquid:methods, Cross-Linking Reagents:chemistry, Cytochrome P450 Family 2, Cytochromes b5:chemistry, Dimerization, Electrons, Mass Spectrometry:methods, Microsomes.   

OBJECTIVES: The cytochrome P450 (P450) and cytochrome b5 are membrane hemoproteins composing together with flavoprotein NADPH:P450 reductase a mixed function oxidase (MFO) system. The knowledge of the interaction between P450 and its redox partners within a MFO system is fundamental to understand P450 reaction mechanism, an electron transport from its redox partner and also detoxification of xenobiotics and/or metabolism of endogenous substrates with all positive or negative aspects for organisms.

METHODS: The chemical cross-linking by soluble carbodiimide (EDC) in combination with the liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) has been employed to characterize the contact surface regions involved in the transient interaction between two catalytic domains of P450 2B4 and cytochrome b5.

RESULTS: The cross-linking reaction was accomplished in an equimolar catalytic complex of P450 2B4:cytochrome b5 and the covalent hetero-dimers detected on SDS-PAGE electrophoresis were analyzed (after in gel trypsin digestion) using LC-HRMS to identify cross-linked amino-acid residues. The computed in silico models of P450 2B4:cytochrome b5 complex using amino-acids participating in cross-links (Asp134, Lys139, Glu424 and Glu439 located on a proximal surface of P450 2B4) suggest interpretation that two different types of cytochrome b5 orientations are present in the studied interaction within a MFO system: the first allowing potential cytochrome b5 electron donation to P450, the second one inducing cytochrome b5 modulation of P450 structural changes.

CONCLUSIONS: The results demonstrated the capability of the used experimental approach to map the interaction between P450 and cytochrome b5 suggesting the formation of multi-meric structures within a MFO system as interpretation of the two observed mutual orientations....

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Comparative Study 2012; 33(Suppl 3): 33-40 PubMed PMID: 23353841 Citation  Keywords:  Benzoflavones:chemistry, Cytochrome P-450 Enzyme Inhibitors, Disulfiram:chemistry, Ditiocarb:chemistry, Ellipticines:chemistry, Enzyme Activation:drug effects, Enzyme Inhibitors:chemistry, Horseradish Peroxidase:antagonists & inhibitors, Humans, Ketoconaz.   

OBJECTIVES: Of several enzymes metabolizing xenobiotics, cytochrome P450 (CYP) and peroxidase enzymes seem to be most important. One of the major challenges in studies investigating metabolism of xenobiotics is to resolve which of these two groups of enzymes is predominant to metabolize individual xenobiotic compounds. Utilization of selective inhibitors of CYP and peroxidase enzymes might be a useful tool to identify the contribution of these enzymes to metabolism of xenobiotics in samples, where both types of enzymes are present. The aim of this study was to investigate specificities of several known CYP inhibitors to these enzymes; whether they inhibit only the CYP enzymes and do not inhibit peroxidases.

METHODS: Since the oxidation of o-anisidine catalyzed by a model peroxidase used, horseradish peroxidase (HRP), is a two-substrate reaction, the inhibition potential of tested chemicals was studied with respect to both peroxidase substrates, o-anisidine and hydrogen peroxide. Initial velocities of o-anisidine oxidation by HRP under various conditions were determined spectrophotometrically.

RESULTS: The CYP inhibitors metyrapone, troleandomycine, disulfiram, sulfaphenazole, quinidine and 1-aminobenzotriazole do not inhibit o-anisidine oxidation catalyzed by HRP. In contrast, ketoconazole, diethyldithiocarbamate, ellipticine, α-naphtoflavone, proadifen SKF525A, piperonylbutoxide, were found to inhibit not only the CYPs, but also the HRP-mediated oxidation of o-anisidine. Interestingly, α-naphtoflavone inhibits oxidation of o-anisidine by HRP with respect to H2O2, but not with respect to o-anisidine. Diethyldithiocarbamate is the most potent peroxidase inhibitor of o-anisidine oxidation with Ki with respect to o-anisidine of 10 μM and Ki with respect to H2O2 of 60 μM, being even the better peroxidase inhibitor than the classical "peroxidase inhibitor" - propyl gallate (Ki with respect to o-anisidine of 60 μM and Ki with respect to H2O2 of 750 μM).

CONCLUSIONS: The results of the present study demonstrate that 1-aminobenzotriazole, a potent inhibitor of various CYP enzymes, seems to be the best candidate suitable for utilization in studies evaluating participation of CYP enzymes in metabolism of xenobiotics in various complex biological materials containing both CYP and peroxidase enzymes. Moreover, precaution to prevent misinterpretation of results is necessary in cases when proadifen SKF525A, piperonylbutoxide, diethyldithiocarbamate, ketoconazole, α-naphtoflavone and ellipticine are used in similar studies (as CYP inhibitors in various complex biological materials containing both CYP and peroxidase enzymes), since these chemicals can except of CYP enzymes inhibit also peroxidase-mediated reactions....

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Journal Article 2012; 33(Suppl 3): 25-32 PubMed PMID: 23353840 Citation  Keywords:  Amino Acid Sequence, Aristolochia:chemistry, Aristolochic Acids:adverse effects, Aryl Hydrocarbon Hydroxylases:chemistry, Catalytic Domain:drug effects, Computer Simulation, Cytochrome P-450 CYP1A1:chemistry, Cytochrome P-450 CYP1A2:chemistry, Cytochrome .   

OBJECTIVES: The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Human cytochrome P450 (CYP) 1A1 and 1A2 enzymes were found to be responsible for the AAI reductive activation to form AAI-DNA adducts, while its structurally related analogue, CYP1B1 is almost without such activity. However, knowledge of the differences in mechanistic details of CYP1A1-, 1A2-, and 1B1- mediated reduction is still lacking. Therefore, this feature is the aim of the present study.

METHODS: Molecular modeling capable of evaluating interactions of AAI with the active site of human CYP1A1, 1A2 and 1B1 under the reductive conditions was used. In silico docking, employing soft-soft (flexible) docking procedure was used to study the interactions of AAI with the active sites of these human enzymes.

RESULTS: The predicted binding free energies and distances between an AAI ligand and a heme cofactor are similar for all CYPs evaluated. AAI also binds to the active sites of CYP1A1, 1A2 and 1B1 in similar orientations. The carboxylic group of AAI is in the binding position situated directly above heme iron. This ligand orientation is in CYP1A1/1A2 further stabilized by two hydrogen bonds; one between an oxygen atom of the AAI nitro-group and the hydroxyl group of Ser122/Thr124; and the second bond between an oxygen atom of dioxolane ring of AAI and the hydroxyl group of Thr497/Thr498. For the CYP1B1:AAI complex, however, any hydrogen bonding of the nitro-group of AAI is prevented as Ser122/Thr124 residues are in CYP1B1 protein replaced by hydrophobic residue Ala133.

CONCLUSION: The experimental observations indicate that CYP1B1 is more than 10× less efficient in reductive activation of AAI than CYP1A2. The docking simulation however predicts the binding pose and binding energy of AAI in the CYP1B1 pocket to be analogous to that found in CYP1A1/2. We believe that the hydroxyl group of S122/T124 residue, with its polar hydrogen placed close to the nitro group of the substrate (AAI), is mechanistically important, for example it could provide a proton required for the stepwise reduction process. The absence of a suitable proton donor in the AAI-CYP1B1 binary complex could be the key difference, as the nitro group is in this complex surrounded only by the hydrophobic residues with potential hydrogen donors not closer than 5 Å....

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Journal Article 2012; 33(Suppl 3): 3-7 PubMed PMID: 23353837 Citation  Keywords:  Amaranthus:chemistry, Crops, Agricultural:chemistry, Health Promotion, Humans, Nutritive Value,.   

: The article deals with the importance and advantages of the amaranth plant - a genus of herbs of the family Amarantaceae. Amaranth, highly nutritional pseudocereal and traditional american crop has good food potential value. Amaranth grain doesn't contain gluten. The high content of quality protein and unsaturated fatty acids is one of its advantages. It is also a carrier of a very valuable fibre and good source of squalene.

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Journal Article 2012; 33(Suppl 2): 102-107 PubMed PMID: 23183520 Citation  Keywords:  Adult, Cardiovascular Diseases:epidemiology, Cross-Sectional Studies, Female, Follow-Up Studies, Humans, Logistic Models, Male, Middle Aged, Multivariate Analysis, Prevalence, Retrospective Studies, Risk Factors, Risk Reduction Behavior, Smoking Cessation.   

OBJECTIVES: Smoking cessation is an essential part of cardiovascular disease (CVD) prevention. At the Center for Tobacco-Dependent (CTD), clients are screened to identify and reduce cardiovascular (CV) risk factors. In our study we have focused on the role of the CTD in reducing global CV risk.

METHODS: 1,334 CTD patients aged 25-64 years (52.2% men, mean age 44±12 years, Fagerström Test for Cigarette Dependence 6±2) were included in a retrospective cross-sectional survey. Medical history, blood samples and physical examination were analysed. Blood pressure, weight and exhaled CO were measured at each visit (12-months-follow-up). Patients' CV risk was scored. CO-verified abstinence according to CV risk and prevalence of detected CV risk factors were examined.

RESULTS: Among patients who had attended at least their first visit and a visit after one year, 37.9% (506/1,334) had stopped smoking. Among patients with a SCORE of <5%, the success rate was 44.3% (254/574) and 41.2% for patients at high CV risk (105/255, p=0.41). There was a trend towards a lower success rate among patients with CVD, but this difference was not significant. The smoking cessation rate among low and high CV risk patients at the baseline visit was identical (46.2%, resp. 47.3%, p=0.81). 3.1% (42/1,334) of patients were referred to a specialist for hypertension. 62.5% (223/357), without a prior history, were found to have dyslipidemia.

CONCLUSIONS: High CV risk patients have the same chance to stop smoking as low risk patients....

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