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Journal Article 2012; 33(Suppl 3): 53-59 PubMed PMID: 23353844 Citation  Keywords:  Animals, BALB 3T3 Cells:cytology, Chick Embryo, Chickens, Chorioallantoic Membrane:cytology, Cornea:cytology, Dose-Response Relationship, Drug, Embryo, Nonmammalian:cytology, In Vitro Techniques, Insecticides:toxicity, Irritants:toxicity, Linear Models, M.   

OBJECTIVES: Malathion is generally not classified as toxic. However, the toxicity seems to be species-dependent. Local and systemic toxicity data for birds are rare, but a decrease of wild bird densities in areas where malathion was applied was reported. Aim of the study was to extend knowledge on malathion toxicity on cellular and organ level and to evaluate embryotoxicity and genotoxicity for birds using the chick embryo model HET-CAM.

METHODS: Skin and eye irritation was determined using reconstructed skin and eye cornea tissues and the chorioallantoic membrane of chick embryo to simulate conjunctiva. Cytotoxicity in 3T3 Balb/c fibroblast culture was determined to estimate acute systemic toxicity. Chick embryo model was further employed to evaluate acute embryotoxicity for birds (mortality and genotoxicity). Data were analysed by means of general linear models.

RESULTS: Malathion is not a skin and eye irritant. Cytotoxicity in vitro test provided LD50 value of 616 mg/kg suggesting higher toxic potential than is generally published based on in vivo tests on laboratory rodents. Embryotoxicity studies revealed dose and age dependent mortality of chick embryos. Genotoxicity was identified by means of micronucleus test in erythroid cells isolated from chorioallantois vascular system of chick embryos.

CONCLUSIONS: Using in vitro alternative toxicological methods, a higher toxic potential of malathion was demonstrated than is generally declared. An increased health and environmental hazard may occur in areas with intensive agricultural production. The environmental consequences of delayed effects and embryotoxicity for bird populations in areas exposed to organophosphate insecticides, such as malathion, are obvious....

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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): 16-24 PubMed PMID: 23353839 Citation  Keywords:  Antineoplastic Agents, Phytogenic:pharmacology, Apoptosis:drug effects, Bone Marrow Neoplasms:drug therapy, Brain Neoplasms:drug therapy, Cell Cycle:drug effects, Cell Line, Tumor, Drug Resistance, Neoplasm:drug effects, Drug Synergism, Etoposide:pharmaco.   

OBJECTIVES: Etoposide (Vepesid, VP-16), an inhibitor of topoisomerase II, is a chemotherapeutic drug commonly used for treatment of different types of malignant diseases. By inhibiting the topoisomerase II enzyme activity in cancer cells, this drug leads to DNA damage and subsequently to cell death. In this study, we investigated the effect of this anticancer drug alone and in combination with a histone deacetylase (HDAC) inhibitor, valproic acid (VPA), on a human UKF-NB-4 neuroblastoma cell line.

METHODS: The effects of etoposide and VPA on UKF-NB-4 cells were tested under the normoxic and also the hypoxic (1% O2) cultivation conditions. The cytotoxicity of etoposide and VPA to a UKF-NB-4 neuroblastoma cell line was evaluated with MTT assay. Apoptosis of the cells was analyzed by flow cytometry using an Annexin V and propidium iodide binding method. The effect of etoposide and VPA on the cell cycle distribution was determined by flow cytometric analysis using propidium iodide staining.

RESULTS: The results of the study demonstrate that UKF-NB-4 neuroblastoma cells are sensitive both to etoposide and to VPA. They also indicate that the impact of VPA on cytotoxicity of etoposide in these tumor cells varies depending on the sequence of cultivation of the cells with the drugs. As a suitable sequence of cultivation, with a high rate of suppression of neuroblastoma cell growth was found the preincubation of the cells with etoposide, which was followed by their cultivation with VPA. In contrast, the reversed combination (preincubation of the cells with VPA before their treating with etoposide) did not give any increase in etoposide cytotoxicity. The effect of such combined treatment can be explained by measuring the cell cycle distribution, which shows that both etoposide and VPA change the cell cycle phase distribution.

CONCLUSION: Etoposide and VPA were found as cycle phase specific drugs that are cytotoxic to human UKF-NB-4 neuroblastoma cells used either as single drugs or both together. However, whereas VPA might sensitize the cells to etoposide, inappropriate sequence of cultivation of the cells with VPA can decrease the etoposide cytotoxic efficacy. The results found here warrant further studies of combined treatment of neuroblastoma cells with etoposide with HDAC inhibitors and may help in the design of new protocols geared to the treatment of high risk neuroblastomas....

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Journal Article 2012; 33(Suppl 3): 8-15 PubMed PMID: 23353838 Citation  Keywords:  Aerobiosis:physiology, Air Pollutants:pharmacokinetics, Anaerobiosis:physiology, Animals, Benz(a)Anthracenes:pharmacokinetics, Carcinogens:pharmacokinetics, Cytochrome P-450 Enzyme System:metabolism, Cytochrome-B(5) Reductase:genetics, DNA Adducts:metabol.   

OBJECTIVES: 2-Nitrobenzanthrone (2-NBA) has recently been detected in ambient air particulate matter. Its isomer 3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. Understanding which enzymes are involved in metabolism of these toxicants is important in the assessment of individual susceptibility. Here, metabolism of 2-NBA and 3-NBA by rat and mouse hepatic microsomes containing cytochromes P450 (CYPs), their reductase (NADPH:CYP reductase), and NADH:cytochrome b5 reductase was investigated under anaerobic and aerobic conditions. In addition, using the same microsomal systems, 2-NBA and 3-NBA were evaluated to be enzymatically activated under anaerobic conditions to species generating 2-NBA- and 3-NBA-derived DNA adducts.

METHODS: High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was employed for the separation and characterization of 2-NBA and 3-NBA metabolites formed by hepatic microsomes of rats and mice under the anaerobic and aerobic conditions. Microsomal systems isolated from the liver of the control (untreated) rats and rats pretreated with Sudan I, β-naphthoflavone (β-NF), phenobarbital (PB), ethanol and pregnenolon 16α-carbonitrile (PCN), the inducers of cytochromes P450 (CYP) 1A1, 1A1/2, 2B, 2E1 and 3A, respectively, were used in this study. Microsomes of mouse models, a control mouse line (wild-type, WT) and Hepatic Cytochrome P450 Reductase Null (HRN) mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in their livers, were also employed. To detect and quantify the 2-NBA- and 3-NBA-derived DNA adducts, the 32P postlabeling technique was used.

RESULTS: Both reductive metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), found to be formed predominantly under the anaerobic conditions, and two 3-NBA oxidative metabolites, whose structures have not yet been investigated, were formed by several microsomal systems used in the study. Whereas a 3-NBA reductive metabolite, 3-ABA, was found only in the microsomal systems of control rats, the rats treated with β-NF and PB, and microsomes of WT and HRN mice, all hepatic microsomes tested in the study were capable of activating this carcinogen under the reductive conditions to form DNA adducts. A stability of a reactive intermediate of 3-NBA, N-hydroxy-3-aminobenzanthrone that is formed during 3-NBA reduction to 3-ABA, to form nitrenium (and/or carbenium) ions binding to DNA in individual microsomes as well as binding of these ions to proteins of these microsomes, might be the reasons explaining this phenomenon. In contrast to 3-NBA, its isomer 2-NBA was not metabolized by any of the used enzymatic systems both under the anaerobic and aerobic conditions. Likewise, no DNA adducts were detectable after reaction of 2-NBA in these systems with DNA.

CONCLUSIONS: The results found in this study, the first report on the metabolism of 2-NBA and 3-NBA by rat and mouse hepatic microsomes demonstrate that 3-NBA, in contrast to 2-NBA, is reductively activated to form 3-NBA-derived DNA adducts by these enzymatic systems. NADPH:CYP reductase can be responsible for formation of these DNA adducts in rat livers, while NADH:cytochrome b5 reductase can contribute to this process in livers of HRN mice....

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Comparative Study 2012; 33(Suppl 2): 93-97 PubMed PMID: 23183518 Citation  Keywords:  Adipocytes:cytology, Adult, Cell Differentiation:physiology, Cells, Cultured, Chemokine CCL2:genetics, Chemokine CCL5:genetics, Cytokines:genetics, Gene Expression:physiology, Humans, Interleukin-18:genetics, Interleukin-6:genetics, Interleukin-8:genetics.   

BACKGROUND: Our study focused on the ability of epicardial adipocytes to produce bioactive substances and compare the extent of this production with the production of adipokines in visceral adipocytes, which are well known endocrine cells capable of contributing to the development of atherosclerosis.

MATERIAL AND METHODS: The gene expression of human cytokines (IL-6, IL-8, IL-18, RANTES and MCP-1) and adipokines (leptin and adiponectin) was measured in primary cell lines of epicardial and visceral adipocytes, both in undifferentiated and mature statuses, after a 21-day-long differentiation protocol. Each condition was assayed in triplicate in two independent primary cell lines obtained from two different donors.

RESULTS: The epicardial preadipocytes showed an increased expression of IL-8 (3.25-fold, p<0.05) compared with visceral preadipocytes. The expression of the atheroprotective adiponectin in epicardial preadipocytes was minimal compared with the expression in visceral preadipocytes (p<0.0001). Moreover, the expression of the genes of interest was dependent on the differentiation degree and cell origin. We observed an altered expression of the proinflammatory genes IL-8 (0.016-fold, p<0.01) and MCP-1 (0.19-fold, p<0.05) in differentiated epicardial adipocytes compared with undifferentiated adipocytes. The epicardial adipocytes showed an increased expression of IL-6 (8.13-fold, p<0.05) compared with the visceral adipocytes.

CONCLUSION: Our results suggest that epicardial adipocytes substantially differ from visceral adipocytes and might locally contribute to the pathogenesis of coronary atherosclerosis....

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