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  • 1
    Keywords: MOUSE ; FUNCTIONAL GENOMICS ; DRUG ; genomic ; E ; BIOACTIVATION ; anticancer drug ; ellipticine ; genomics ; cytochrome P450 ; hepatic
    Type of Publication: Book chapter
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  • 2
    Keywords: CANCER ; human ; MODEL ; MODELS ; DNA adducts ; SITE ; SITES ; ENZYMES ; TISSUE ; RESOLUTION ; ACTIVATION ; LIGAND ; DNA ; REDUCTION ; DNA ADDUCT FORMATION ; TISSUES ; BINDING ; ACID ; PATTERNS ; HUMANS ; ASSAY ; ACTIVE-SITE ; DNA-BINDING ; METABOLIC-ACTIVATION ; ADDUCTS ; ORIENTATION ; BINDS ; aristolochic acid ; BALKAN ENDEMIC NEPHROPATHY ; CHINESE HERBS NEPHROPATHY ; P-32 POSTLABELING ANALYSIS ; DNA-ADDUCTS ; RECOMBINANT ; CRYSTALLOGRAPHIC STRUCTURE ; urothelial cancer ; CARCINOGEN ; REDUCTASE ; interaction ; development ; IRON ; ADDUCT ; ENZYME ; DNA ADDUCT ; P-32-postlabeling ; docking ; human cytochromes P450 ; computer modeling ; cytochromes P450 1A1 and 1A2 ; DNA binding ; NADPH ; reductive activation
    Abstract: Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Using the P-32-postlabeling assay we showed that AAI is activated by human recombinant cytochrome P450 (CYP) 1A1, CYPIA2 and NADPH:CYP reductase to species generating DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N-6-yl)aristolactam I, 7-(deoxyguanosin-N-2-yl) aristolactam I and 7-(deoxyadenosin-N-6-yl)aristolactam II were identified as AA-DNA adducts formed from AAI by the enzymes. The formation of these AA-derived DNA adducts indicates that all the human enzymes reduce the nitro group of AAI to the putative reactive cyclic nitrenium ion responsible for adduct formation. The concentrations of AAI required for its half-maximum DNA binding were 38,65 and 126 mu M AAI for reductive activation by human CYP1A2, CYP1A1 and NADPH:CYP reductase, respectively. CYP1A1 and 1A2 homology modeling followed by docking of AAI to the CYP1A1 and 1A2 active centers was utilized to explain the potential of these enzymes to reduce AAI. Models of human CYP1A1 and 1A2 were constructed on the basis of the crystallographic structure of truncated mammalian CYP enzymes, CYP2B4, 2C5, 2C8, 2C9 and 3A4. The in silico docking of AAI to the active sites of CYP1A1I and 1A2 indicates that AAI binds as an axial ligand of the heme iron and that the nitro group of AAI is in close vicinity to the heme iron of CYPIA2 in an orientation allowing the efficient reduction of this group observed experimentally. The orientation of AAI in the active centre of CYP1A1 however causes an interaction of the heme iron with both the nitro- and the carboxylic groups of AAI. This observation explains the lower reductive potential of CYP1A1 for AAI than CYP1A2, detected experimentally. (c) 2005 Elsevier Ireland Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16125300
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  • 3
    Keywords: EXPRESSION ; IN-VITRO ; INHIBITOR ; AGENTS ; human ; INHIBITION ; MODEL ; MODELS ; VITRO ; SYSTEM ; SYSTEMS ; DNA adducts ; liver ; ENZYMES ; DRUG ; METABOLISM ; DNA ; RAT ; animals ; RATS ; SUSCEPTIBILITY ; METABOLITES ; HUMANS ; ADDUCTS ; OXIDATION ; cytochrome P450 ; INHIBITORS ; DNA-ADDUCTS ; ENZYME ; DNA ADDUCT ; comparative study ; pharmacology ; Male ; animal ; magnetic resonance spectroscopy ; animal model ; CYP ; anticancer drug ; ANTICANCER DRUG ELLIPTICINE ; ANTICANCER-DRUG ; ellipticine ; drug effects ; DNA intercalation ; antagonists & inhibitors ; Antineoplastic Agents ; Cytochrome P-450 Enzyme System ; Ellipticines ; enzyme inhibitors ; Hydroxylation ; Microsomes,Liver ; Oxidation-Reduction ; Rabbits ; Rats,Wistar ; Species Specificity
    Abstract: Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellipticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only rat microsomes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N(2)-oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in expression and catalytic activities of individual CYPs in livers are the cause of these metabolic differences. Formation of 7-hydroxy- and 9-hydroxyellipticine was attributable to CYP1A in microsomes of all species. However, production of 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, the metabolites generating DNA adducts, was attributable to the orthologous CYPs only in rats and humans. CYP3A predominantly generates these metabolites in rat and human microsomes, while CYP2C3 activity prevails in microsomes of rabbits. The results underline the suitability of rat species as a model to evaluate human susceptibility to ellipticine
    Type of Publication: Journal article published
    PubMed ID: 17197724
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  • 4
    Keywords: EXPRESSION ; LUNG ; liver ; PROTEIN ; DRUG ; EFFICIENCY ; TISSUE ; kidney ; 3-nitrobenzanthrone ; DNA ADDUCT FORMATION ; ENVIRONMENTAL CONTAMINANT 3-NITROBENZANTHRONE ; metabolic activation ; INDUCTION ; RAT ; BODY-WEIGHT ; RATS ; TISSUES ; NO ; IDENTIFICATION ; RAT-LIVER ; HUMAN ACETYLTRANSFERASES ; METABOLIC-ACTIVATION ; POLLUTANT 3-NITROBENZANTHRONE ; POLYMERASE-CHAIN-REACTION ; CARCINOGENS ; CHAIN-REACTION ; BODY ; protein expression ; ANTICANCER DRUGS ; DT-DIAPHORASE ; AGENT ; BODIES ; CHEMISTRY ; CHAIN ; INCREASE ; polymerase chain reaction ; WEIGHT ; body weight ; REAL-TIME ; SUBSTRATE ; LEVEL ; ENZYME ; DRUGS ; Male ; reductive activation ; lungs ; anticancer drug ; ellipticine ; enzymatic ; QUINONE OXIDOREDUCTASE ; CYTOCHROMES P450 ; correlates ; ANTIOXIDANT-RESPONSE-ELEMENT ; enzyme induction ; NAD(P)H : quinone oxidoreductase
    Abstract: The antineoplastic agent ellipticine was investigated for its ability to induce the biotransformation enzyme NAD(P) H:quinone oxidoreductase (DT-diaphorase, EC 1.6.99.2) in male Wistar rats. Using the real-time polymerase chain reaction, the levels of NAD(P) H:quinone oxidoreductase mRNA were determined in livers, kidneys and lungs of rats treated intraperitoneally with ellipticine (40 mg/kg body weight) and of control (untreated) rats. Cytosolic fractions were isolated from the same tissues of control and ellipticine-treated rats and tested for NAD(P) H:quinone oxidoreductase protein expression and its enzymatic activity. The results demonstrate that ellipticine is a potent inducer of NAD(P) H:quinone oxidoreductase in rat livers and kidneys, while no induction of this enzyme was detectable in rat lungs. The increase in levels of NAD(P) H:quinone oxidoreductase mRNA correlates with the increase in expression of its protein and enzymatic activity, measured with menadione and 3-nitrobenzanthrone as substrates. The results, the identification of the potential of ellipticine to induce NAD(P) H: quinone oxidoreductase, suggest that this drug is capable of modulating biological efficiencies of the toxicants and/or drugs that are reductively metabolized by this enzyme
    Type of Publication: Journal article published
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  • 5
    Keywords: human ; 3-aminobenzanthrone ; 3-nitrobenzanthrone ; RAT ; POLLUTANT 3-NITROBENZANTHRONE ; HUMAN METABOLITE
    Type of Publication: Journal article published
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  • 6
    Keywords: IN-VITRO ; human ; IN-VIVO ; LUNG ; MODEL ; VITRO ; DNA adducts ; liver ; ENZYMES ; METABOLISM ; MICE ; ACTIVATION ; DNA ; kidney ; DNA ADDUCT FORMATION ; LIVER-MICROSOMES ; RAT ; P-32-postlabelling ; BINDING ; MOUSE ; PATTERNS ; DNA-BINDING ; METABOLIC-ACTIVATION ; OXIDATION ; cytochrome P450 ; AGENT ; BODIES ; PATTERN ; WEIGHT ; LEVEL ; pharmacology ; USA ; LOSSES ; PROSTAGLANDIN-H SYNTHASE ; anticancer drug ; ellipticine ; ENVIRONMENTAL-POLLUTANT 3-NITROBENZANTHRONE ; peroxidase ; DETERMINES SUSCEPTIBILITY ; XENOBIOTIC-METABOLISM
    Abstract: Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by P-32-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study. (c) 2007 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17976674
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  • 7
    Keywords: METABOLISM ; ACTIVATION ; DNA ; MECHANISM ; SUDAN-I ; HPLC ; BINDING ; MAGNETIC-RESONANCE ; METABOLITES ; MOLECULE ; SPECTROSCOPY ; TARGET ; PERFORMANCE ; NMR ; SWEDEN ; ELECTRON ; CHROMATOGRAPHY ; LIQUID-CHROMATOGRAPHY ; OXIDATION ; LAYER ; detoxication ; PRODUCTS ; CARCINOGEN ; NUCLEAR-MAGNETIC-RESONANCE ; ENZYME ; methods ; CYTOCHROME-P450 1A1 ; NUCLEAR ; MASS ; SEPARATION ; peroxidase ; high performance liquid ; ONE-ELECTRON ; 1-PHENYLAZO-2-HYDROXYNAPHTHALENE ; BENZENEDIAZONIUM ION ; mechanism of oxidation ; Sudan I ; TRANSFER-RIBONUCLEIC-ACID
    Abstract: OBJECTIVES: The aim of the study was to examine oxidation of carcinogenic Sudan I by peroxidase and characterize the structure of its two major peroxidase-mediated metabolites. Another target of the study was to evaluate a mechanism of this oxidation. METHODS: Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) with ultraviolet (UV) and visible (VIS) detection was employed for the separation of Sudan I metabolites formed by peroxidase. UV/VIS, and mass- spectroscopy as well as nuclear magnetic resonance (NMR) were used to characterize structures of two major Sudan I metabolites. RESULTS: Peroxidase oxidizes Sudan I by a one electron oxidation to eight products. Two major Sudan I metabolites were isolated by TLC on silica gel and HPLC and structurally characterized. The major product formed during the Sudan I oxidation by peroxidase is Sudan I metabolite M-2, which corresponds to a Sudan I dimer molecule. The second major metabolite (M-1) is the product of secondary, enzyme independent reactions, being formed from the Sudan I dimer that lost the benzenediazonium moiety. CONCLUSIONS: The data are the first report on structural characterization of Sudan I metabolites formed by its oxidation with peroxidase
    Type of Publication: Journal article published
    PubMed ID: 18987613
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  • 8
    Keywords: CANCER ; ENZYMES ; ACID ; INJURY ; aristolochic acid ; ENZYME ; RENAL INJURY
    Type of Publication: Journal article published
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  • 9
    Keywords: 3-aminobenzanthrone, 3-nitrobenzanthrone, ACTIVATION, ADDUCTS, animals, Benz(a)Anthracenes, biotrans
    Abstract: 3-aminobenzanthrone (3-ABA) is the metabolite of the carcinogenic air pollutant 3-nitrobenzanthrone (3-NBA). 3-ABA was investigated for its ability to induce cytochrome P450 1A1 (CYP1A1) and NAD(P)H:quinone oxidoreductase (NQO1) in kidney and lung of rats, and for the influence of such induction on DNA adduct formation by 3-ABA and 3-NBA. NQO1 is the enzyme that reduces 3-NBA to N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) and CYP1A enzymes oxidize 3-ABA to the same intermediate. When activated by cytosolic and and/or microsomal fractions isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney, both compounds generated the same DNA-adduct pattern, consisting of five adducts. When pulmonary cytosols isolated from rats that had been treated i.p. with 40 mg/kg bw of 3-ABA were incubated with 3-NBA, DNA adduct formation was up to 1.7-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. In contrast, no induction of NQO1 expression by 3-ABA treatment was found in the kidney. Incubations of 3-ABA with renal and pulmonary microsomes of 3-ABA-treated rats led to an increase of up to a 4.5-fold in DNA-adduct formation relative to controls. The stimulation of DNA-adduct formation correlated with a higher protein expression and activity of CYP1A1 induced by 3-ABA. These results show that by inducing lung and kidney CYP1A1 and NQO1, 3-ABA increases its own enzymatic activation as well as that of the environmental pollutant, 3-NBA, thereby enhancing the genotoxic and carcinogenic potential of both compounds
    Type of Publication: Journal article published
    PubMed ID: 19398038
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  • 10
    Keywords: IN-VITRO ; IN-VIVO ; DNA adducts ; METABOLISM ; ACTIVATION ; DNA ADDUCT FORMATION ; PATTERNS ; DNA-BINDING ; aristolochic acid ; BALKAN ENDEMIC NEPHROPATHY ; CHINESE HERBS NEPHROPATHY ; P-32 POSTLABELING ANALYSIS ; DNA-ADDUCTS ; ACETYLTRANSFERASE ; urothelial cancer ; CARCINOGEN ; ADDUCT FORMATION ; DNA ADDUCT ; sulfotransferase ; P-32-postlabeling ; molecular modeling ; reductive activation ; PROSTAGLANDIN-H SYNTHASE ; CYTOCHROMES P450 1A1 ; NAD(P)H:quinone oxidoreductase ; HUMAN CANCER HAZARD ; HUMAN CARCINOGEN
    Abstract: OBJECTIVES: Ingestion of aristolochic acid (AA) is associated with development of urothelial tumors linked with aristolochic acid nephropathy, and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. Aristolochic acid I (AAI), the major toxic component of AA, is more toxic than its demethoxylated derivate AAII. A different enzymatic conversion of both carcinogens might be one of the reasons explaining this feature. Therefore, the present study has been designed to compare efficiency of human NAD(P)H:quinone oxidoreductase (NQO1) and phase II enzymes such as sulfotransferases (SULTs) and N,O-acetyltransferases (NATs) to activate AAI and AAII in vitro. In addition, to investigate the molecular mechanisms of AAI and AAII reduction by human NQO1, molecular modeling was used to compare interactions of AAI and AAII with the active site of this enzyme. METHODS: DNA adduct formation by AAI and AAII was investigated by the nuclease P1 version of the 32P-postlabeling method. In silico docking, employing soft-soft (flexible) docking procedure, was used to study the interactions of AAI and AAII with the active site of human NQO1. RESULTS: Human NQO1 activated AAI and AAII, generating DNA adduct patterns reproducing those found in several species including human exposed to these compounds. These results demonstrate that NQO1 is capable of reducing both AAs to reactive species binding to DNA. However, concentrations required for half-maximum DNA binding mediated by NQO1 were higher for AAII (158 microM) than for AAI (17 microM). One of the reasons causing this phenomenon is a lower efficiency of NQO1 to reduce AAII than AAI we found in this work; although both AAI and AAII are bound with similar binding affinities to the NQO1 active site, the binding orientation of AAII in the active site of NQO1 does not favor the effective reduction of its nitro group. Because reduced nitro-aromatics are often further activated by SULTs or NATs, their roles in AAI and AAII activation were investigated. Our results indicate that phase II reactions do not stimulate the bioactivation of AAs; neither enzymes present in human hepatic cytosols nor human SULT1A1, 1A2, 1A3, 1E, or 2A nor NAT1 or NAT2 further enhanced DNA adduct formation by AAs. In contrast, human SULT1A1, 1A2 and 1A3 as well as NAT1 and NAT2 enzymes even inhibited NQO1-mediated bioactivation of AAII. Therefore, under the in vitro conditions used, DNA adducts arise by enzymatic reduction of AAs through the formation of N-hydroxyaristolactams that are spontaneously decomposed to the reactive species forming DNA adducts. CONCLUSION: The results found in this study emphasize the importance of NQO1 in the metabolic activation of AAI and AAII and provide the evidence that initial nitroreduction is the rate limiting step in their activation. This enzyme is more effective in activation of AAI relative to AAII, which might contribute to its lower binding to DNA found both in vitro and in vivo, Moreover, inhibition effects of conjugation reactions on AAII activation might further contribute to its decreased capability of forming DNA adducts and its lower toxicity comparing with AAI.
    Type of Publication: Journal article published
    PubMed ID: 22167209
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