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  • 1
    Keywords: IN-VITRO ; INHIBITOR ; IN-VIVO ; VITRO ; DNA adducts ; liver ; ENZYMES ; METABOLISM ; ACTIVATION ; DNA ; kidney ; MECHANISM ; DNA ADDUCT FORMATION ; metabolic activation ; LIVER-MICROSOMES ; RAT ; AROMATIC-AMINES ; RATS ; METABOLITES ; IDENTIFICATION ; HUMANS ; ASSAY ; URINARY-BLADDER ; NUCLEOTIDES ; ADDUCTS ; RISK ASSESSMENT ; OXIDATION ; BINDS ; rodent ; CYTOCHROME-P-450 ; INHIBITORS ; DNA-ADDUCTS ; CAPACITY ; CARCINOGEN ; SELECTIVE INHIBITORS ; ADDUCT ; HEMOGLOBIN ADDUCTS ; DNA ADDUCT ; P-32-postlabeling ; rodents ; human cytochromes P450 ; o-anisidine ; TOBACCO-SPECIFIC NITROSAMINES
    Abstract: 2-methoxyaniline (o-anisidine) is an industrial and environmental pollutant and a bladder carcinogen for rodents. The mechanism of its carcinogenicity was investigated with 2 independent methods, P-32-postlabeling and C-14-labeled o-anisidine, to show that o-anisidine binds covalently to DNA in vitro after its activation by human hepatic microsomes. We also investigated the capacity of o-anisidine to form DNA adducts in vivo. Rats were treated i.p. with o-anisidine (0.15 mg/kg daily for 5 days) and DNA from several organs was analyzed by P-32-postlabeling. Two o-anisidine-DNA adducts, identical to those found in DNA incubated with o-anisidine and human microsomes in vitro, were detected in urinary bladder (4.1 adducts per 107 nucleotides), the target organ, and, to a lesser extent, in liver, kidney and spleen. These DNA adducts were identified as deoxyguanosine adducts derived from a metabolite of o-anisidine, N-(2-methoxyphenyl)hydroxylamine. This metabolite was identified in incubations with human microsomes. With 9 human hepatic microsomal preparations, we identified the specific CYP catalyzing the formation of the o-anisidine metabolites by correlation studies and by examining the effects of CYP inhibitors. On the basis of these analyses, oxidation of o-anisidine was attributed mainly to CYP2E1. Using recombinant human CYP (in Supersomes) and purified CYPs, the participation of CYP2E1 in o-anisidine oxidation was confirmed. In Supersomes, CYP1A2 was even more efficient in oxidizing o-anisidine than CYP2E1, followed by CYP2B6, 1A1, 2A6, 2D6 and 3A4. The results, the first report on the potential of the human microsomal CYP enzymes to activate o-anisidine, strongly suggest a carcinogenic potential of this rodent carcinogen for humans. (c) 2005 Wiley-Liss, Inc
    Type of Publication: Journal article published
    PubMed ID: 15828049
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  • 2
    Keywords: ACTIVATION ; LIVER-MICROSOMES ; detoxication ; BALKAN ENDEMIC NEPHROPATHY ; urothelial cancer ; RISK-FACTOR ; ARISTOLACTAM-DNA ADDUCTS ; CYTOCHROMES P450 1A1/2 ; VITAMIN-K METABOLISM ; NAD(P)H/QUINONE OXIDOREDUCTASE
    Abstract: Aristolochic acid I (AAI) is the major toxic component of the plant extract AA, which leads to the development of nephropathy and urothelial cancer in human. Individual susceptibility to AAI-induced disease might reflect variability in enzymes that metabolise AAI. In vitro NAD(P)H: quinone oxidoreductase (NQO1) is the most potent enzyme that activates AAI by catalyzing formation of AAI-DNA adducts, which are found in kidneys of patients exposed to AAI. Inhibition of renal NQO1 activity by dicoumarol has been shown in mice. Here, we studied the influence of dicoumarol on metabolic activation of AAI in Wistar rats in vivo. In contrast to previous in vitro findings, dicoumarol did not inhibit AAI-DNA adduct formation in rats. Compared with rats treated with AAI alone, 11- and 5.4-fold higher AAI-DNA adduct levels were detected in liver and kidney, respectively, of rats pretreated with dicoumarol prior to exposure to AAI. Cytosols and microsomes isolated from liver and kidney of these rats were analysed for activity and protein levels of enzymes known to be involved in AAI metabolism. The combination of dicoumarol with AAI induced NQO1 protein level and activity in both organs. This was paralleled by an increase in AAI-DNA adduct levels found in ex vivo incubations with cytosols from rats pretreated with dicoumarol compared to cytosols from untreated rats. Microsomal ex vivo incubations showed a lower AAI detoxication to its oxidative metabolite, 8-hydroxyaristolochic acid (AAIa), although cytochrome P450 (CYP) 1A was practically unchanged. Because of these unexpected results, we examined CYP2C activity in microsomes and found that treatment of rats with dicoumarol alone and in combination with AAI inhibited CYP2C6/11 in liver. Therefore, these results indicate that CYP2C enzymes might contribute to AAI detoxication.
    Type of Publication: Journal article published
    PubMed ID: 24598128
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  • 3
    Keywords: IN-VIVO ; METABOLIC-ACTIVATION ; POLYCYCLIC AROMATIC-HYDROCARBONS ; MASS-SPECTROMETRY ; LIQUID-CHROMATOGRAPHY ; DNA-ADDUCTS ; ANTICANCER DRUG ELLIPTICINE ; P450 REDUCTASE ; NADPH-CYTOCHROME-P-450 REDUCTASE ; P450-P450 INTERACTIONS
    Abstract: In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b5, and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was approximately 1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b5 participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.
    Type of Publication: Journal article published
    PubMed ID: 24530354
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  • 4
    Keywords: INHIBITOR ; human ; MODEL ; MODELS ; SYSTEM ; liver ; ENZYMES ; METABOLISM ; DNA ; LIVER-MICROSOMES ; RAT ; SUDAN-I ; AROMATIC-AMINES ; ASSAY ; mass spectrometry ; MODULATION ; EFFICIENT ; MASS-SPECTROMETRY ; CYTOCHROME-P-450 ; INHIBITORS ; CHEMISTRY ; RE ; SUBSTRATE ; HEMOGLOBIN ADDUCTS ; ENZYME ; MASS ; rodents ; USA ; FREE-RADICALS ; TOBACCO-SPECIFIC NITROSAMINES ; animal ; GENOTOXIC MECHANISM
    Abstract: We investigated the ability of hepatic microsomes from rat and rabbit to metabolize 2-methoxyaniline (o-anisidine), an industrial and environmental pollutant and a bladder carcinogen for rodents. Using HPLC combined with electrospray tandem mass spectrometry, we determined that o-anisidine is oxidized by microsomes of both species to N-(2-methoxyphenyl)hydroxylamine, o-aminophenol, and one additional metabolite, the exact structure of which has not been identified as yet. N-(2-Methoxyphenyl)hydroxylamine is either further oxidized to 2-methoxynitrosobenzene (o-nitrosoanisole) or reduced to parental o-anisidine, which can be oxidized again to produce o-aminophenol. To define the role of microsomal cytochromes P450 (P450) in o-anisidine metabolism, we investigated the modulation of this metabolism by specific inducers and selective inhibitors of these enzymes. The results of the studies suggest that o-anisidine is a promiscuous substrate of P450s of rat and rabbit liver; because P450s of 1A, 2B, 2E, and 3A subfamilies metabolize o-anisidine in hepatic microsomes of both studied species. Using purified enzymes of rat and rabbit (P450s 1A1, 1A2, 2B2, 2B4, 2E1, 2C3, 3A1, and 3A6), reconstituted with NADPH:P450 reductase, the ability of P450s 1A1, 1A2, 2B2, 2B4, 2E1, and 3A6 to metabolize o-anisidine was confirmed. In the reconstituted P450 system, rabbit P450 2E1 was the most efficient enzyme metabolizing o-anisidine. The data demonstrate the participation of different rat and rabbit P450s in o-anisidine metabolism and indicate that both experimental animal species might serve as suitable models to mimic the fate of o-anisidine in human
    Type of Publication: Journal article published
    PubMed ID: 18624415
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  • 5
    Abstract: OBJECTIVES: Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of activity of several cytochromes P450 (CYP). To elucidate the mechanism of such modulations it is necessary to evaluate not only the effect of native cyt b5, but also that of apo-cyt b5. To prepare apo-cyt b5, heme transfer from native cyt b5 to a protein with higher affinity toward the heme, the horse heart apo-myoglobin, was utilized. METHODS: Butanone extraction was employed to prepare apo-myoglobin. Apo-cyt b5 was separated from myoglobin by chromatography on DEAE-Sepharose. Mass spectrometry was utilized to characterize proteins eluted from DEAE- Sepharose. RESULTS: The prepared apo-myoglobin was incubated with the cyt b5 at pH 4.2 that is the optimal pH for heme transfer from cyt b5 into apo-myoglobin. The apo-cyt b5 protein was separated from myoglobin present in the reaction mixture by chromatography on a column of DEAE-Sepharose. Using such a procedure, 16% yield of apo-cyt b5 that did not contain any heme in its molecule was obtained from the native rabbit cyt b5. Oxidized and reduced forms of the apo-b5 reconstituted with heme exhibit the same absorbance spectra as native cyt b5. The prepared apo-cyt b5 reconstituted with heme can receive electrons from NADPH:CYP reductase. CONCLUSION: A biologically active apo-cyt b5 was prepared using transfer of heme from cyt b5 to horse heart apo-myoglobin by the procedure described here.
    Type of Publication: Journal article published
    PubMed ID: 20027148
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  • 6
    Keywords: METABOLIC-ACTIVATION ; BALKAN ENDEMIC NEPHROPATHY ; CHINESE HERBS NEPHROPATHY ; DT-DIAPHORASE ; urothelial cancer ; ANTICANCER DRUG ELLIPTICINE ; NAD(P)H-QUINONE OXIDOREDUCTASE ; CYTOCHROMES P450 1A1 ; ARISTOLACTAM-DNA ADDUCTS ; ARISTOLOCHIC ACID I
    Abstract: OBJECTIVES: Dicoumarol is known to act as an inhibitor of NAD(P)H: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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
    Type of Publication: Journal article published
    PubMed ID: 25638376
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  • 7
    Abstract: Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. In this study human recombinant CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5) were expressed in Supersomes together with their reductases, NADPH:CYP oxidoreductase, epoxide hydrolase and cytochrome b5 , to investigate BaP metabolism. Human CYPs produced up to eight BaP metabolites. Among these, BaP-7,8-dihydrodiol and BaP-9-ol, which are intermediates in BaP-derived DNA adduct formation, were mainly formed by CYP1A1 and 1B1, and to a lesser extent by CYP2C19 and 3A4. BaP-3-ol, a metabolite that is a 'detoxified' product of BaP, was formed by most human CYPs tested, although CYP1A1 and 1B1 produced it the most efficiently. Based on the amounts of the individual BaP metabolites formed by these CYPs and their expression levels in human liver, we determined their contributions to BaP metabolite formation in this organ. Our results indicate that hepatic CYP1A1 and CYP2C19 are most important in the activation of BaP to BaP-7,8-dihydrodiol, whereas CYP2C19, 3A4, and 1A1 are the major enzymes contributing to the formation of BaP-9-ol. BaP-3-ol is predominantly formed by hepatic CYP3A4, while CYP1A1 and 2C19 are less active. Environ. Mol. Mutagen. 57:229-235, 2016. (c) 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.
    Type of Publication: Journal article published
    PubMed ID: 26919089
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  • 8
    Abstract: ABSTRACT: Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. Here we investigated the efficiencies of rat hepatic microsomes and rat recombinant CYP1A1 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b 5 reductase, epoxide hydrolase and/or cytochrome b 5 in Supersomes to metabolize this carcinogen. We also studied the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b 5 reductase, to mediate BaP metabolism in these systems. Up to eight BaP metabolites and two DNA adducts were generated by the systems, both in the presence of NADPH and NADH. Among BaP metabolites, BaP-9,10-dihydrodiol, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, and a metabolite of unknown structure were formed by hepatic microsomes and rat CYP1A1. One of two DNA adducts formed by examined enzymatic systems (rat hepatic microsomes and rat CYP1A1) was characterized to be 10-(deoxyguanosin-N 2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N 2-BPDE), while another adduct has similar chromatographic properties on polyethylaneimine-cellulose thin layer chromatography to a guanine adduct derived from reaction with 9-hydroxy-BaP-4,5-oxide. In the presence of either of the reductase cofactors tested, NADPH or NADH, cytochrome b 5 stimulated CYP1A1-mediated formation of both BaP-DNA adducts. The results demonstrate that NADH can act as a sole electron donor for both the first and the second reduction of CYP1A1 during its reaction cycle catalyzing oxidation of BaP, and suggest that the NADH:cytochrome b 5 reductase as the NADH-dependent reductase might substitute POR in this enzymatic system. GRAPHICAL ABSTRACT:
    Type of Publication: Journal article published
    PubMed ID: 27110038
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  • 9
    Keywords: PROTEINS ; DNA ADDUCT FORMATION ; SUDAN-I ; MASS-SPECTROMETRY ; glycosylation ; free radicals ; ANTICANCER DRUG ELLIPTICINE ; ellipticine ; (glyco)protein modification by free radicals ; ALPHA(1)-ACID GLYCOPROTEIN ; COVALENT BINDING DATA ; HORSERADISH-PEROXIDASE-C ; HRP ; N-GLYCOSYLATION ; RIBONUCLEASE-B ; Sudan I (1-phenylazo-2-naphthol) ; TAMM-HORSFALL GLYCOPROTEIN
    Abstract: Free radicals generated during peroxidase-catalyzed oxidation of two xenobiotics, carcinogenic Sudan I and an anticancer agent ellipticine, easily attack unmodified proteins but not glycoproteins. A significant inverse correlation between the extent of glycosylation of proteins and the degree of binding of Sudan I or ellipticine radicals to these proteins was observed, whereby the protection only occurs if oligosaccharides are covalently bound to the proteins. No influence of any other variables was found and further confirmed by experiments with proteins containing identical polypeptide chains differing only by the absence (ribonuclease A) or the presence (ribonuclease B) of a single oligosaccharide. The free radicals that are subject of this study did not react with the oligosaccharides because higher levels of the corresponding dimers, reaction products of the radicals, were found in presence of highly glycosylated proteins. The results indicate that carbohydrates protect polypeptides against modification by free radicals derived from toxic xenobiotics and provide passive shielding of the protein moiety
    Type of Publication: Journal article published
    PubMed ID: 20616208
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  • 10
    Keywords: METABOLISM ; HPLC ; benzo[a]pyrene
    Abstract: A simple and sensitive method was developed to separate the carcinogenic polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), and six of its oxidation metabolites generated by rat hepatic microsomes enriched with cytochrome P450 (CYP) 1A1, by high pressure liquid chromatography (HPLC). The HPLC method, using an acetonitrile/water gradient as mobile phase and UV detection, provided appropriate separation and detection of both mono- and di-hydroxylated metabolites of BaP as well as BaP diones formed by rat hepatic microsomes and the parental BaP. In this enzymatic system, 3-hydroxy BaP, 9-hydroxy BaP, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-9,10-dihydrodiol and BaP-dione were generated. Among them the mono-hydroxylated BaP metabolite, 3-hydroxy BaP followed by di-hydroxylated BaP products, BaP-7,8-dihydrodiol and BaP-9,10-dihydrodiol, predominated, while BaP-dione was a minor metabolite. This HPLC method will be useful for further defining the roles of the CYP1A1 enzyme with both in vitro and in vivo models in understanding its real role in activation and detoxification of BaP.
    Type of Publication: Journal article published
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