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  • 1
    Keywords: Germany ; human ; liver ; RISK ; SAMPLE ; SAMPLES ; MONOCLONAL-ANTIBODY ; TISSUE ; DNA ; RISK-FACTORS ; TISSUES ; BREAST ; antibodies ; antibody ; ESCHERICHIA-COLI ; risk factors ; TANDEM MASS-SPECTROMETRY ; DAMAGE ; RISK FACTOR ; NUCLEOTIDES ; DNA-DAMAGE ; MONOCLONAL-ANTIBODIES ; LIQUID-CHROMATOGRAPHY ; LIPID-PEROXIDATION ; OXIDATIVE STRESS ; mutagenesis ; HUMAN BREAST ; HUMAN TISSUES ; MAJOR DNA ADDUCT ; MALONALDEHYDE ; PYRIMIDOPURINONE
    Abstract: The malondialdehyde-modified DNA adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)one (M(1)dG) has been detected in human tissues and is considered to be a promising biomarker for estimating lipid peroxidation-induced DNA damage. With the aim to analyze the M(1)dG in small amounts of DNA (〈 10 lug) and to improve the sensitivity, we have developed an immuno-enriched P-32-postlabeling HPLC method. The main modifications included the following steps: (i) an optimization of the immunoenrichment conditions using a monoclonal antibody (MAb D 10A1), (ii) a single labeling step of the purified M(1)dG 3'-monophosphate to its 5'-monophosphate at pH 6.8, (iii) the addition of O-4-ethylthymidine X-monophosphate as an internal standard, and (iv) a prepurification of the labeled adduct on a polyethyleneimine minicolumn before HPLC analysis. With this protocol, the percent recovery of M(1)dG was found to be similar to70 +/- 20; the detection limit in biological samples was similar to200 amol M(1)dG from 10 mug of DNA, corresponding to 6 adducts/10(9) nucleotides. In conclusion, our modified method shows a high sensitivity and specificity; when applied to human breast and liver tissue samples, background levels of the M(1)dG could be reproducibly detected. This ultrasensitive detection method is thus suitable for applications in human biomonitoring and molecular epidemiology studies
    Type of Publication: Journal article published
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  • 2
    Keywords: human ; liver ; MECHANISM ; SUDAN-I ; HPLC ; CARBON ; MAGNETIC-RESONANCE ; FORM ; PERFORMANCE ; HUMANS ; NMR ; BLADDER ; ADDUCTS ; LIQUID-CHROMATOGRAPHY ; OXIDATION ; DIMER ; rodent ; METABOLITE ; POTENT ; CHEMISTRY ; structure ; NUCLEAR-MAGNETIC-RESONANCE ; COMPOUND ; Urinary bladder
    Abstract: We investigated peroxidase-mediated oxidation of and the formation of the (deoxy)guanosine adduct by 1-phenylazo-2-hydroxynaphthalene (Solvent Yellow 14, Sudan I), a liver and urinary bladder carcinogen for rodents and a potent contact allergen and sensitizer for humans. Using thin layer chromatography (TLC) and/or high performance liquid chromatography (HPLC) combined with mass and/or nuclear magnetic resonance (NMR) spectrometry, we characterized the structures of two major peroxidase-mediated Sudan I metabolites and those of the adducts of (deoxy)guanosine that are formed during Sudan I oxidation. Peroxidase oxidizes Sudan I to radical species that react with another Sudan I radical to form the Sudan I dimer, or in the presence of (deoxy)guanosine, the oxidized Sudan I can attack the exocyclic amino group of guanine, forming the 4-[(deoxy)guanosin-N(2)-yl]Sudan I adduct. The reaction product with a second Sudan I radical results in a dimer where the oxygen 2 radical of Sudan I reacted with carbon 1 in the second Sudan I skeleton. The Sudan I dimer is unstable and decomposes spontaneously to the second oxidation product. This compound consists of the 4-oxo-Sudan I skeleton connected via the oxygen of its 2-hydroxyl group and nitrogen of its azo group with carbon 1 of 2-oxonaphthalene, having a unique spironaphthooxadiazine structure. If (deoxy)guanosine is present during the formation of this Sudan I metabolite, an adduct, in which this Sudan I metabolite is bound to the exocyclic amino group of guanine, is generated. This (deoxy)guanosine adduct is again unstable and decomposes spontaneously to the same adduct that is formed by the direct reaction of oxidized Sudan I, the 4-[(deoxy)guanosin-N(2)-yl]Sudan I adduct. The results presented here are the first structural characterization of Sudan I-(deoxy)guanosine adducts formed during the oxidation of this carcinogen by peroxidase
    Type of Publication: Journal article published
    PubMed ID: 19813759
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  • 3
    Keywords: CANCER ; EXPRESSION ; DISEASES ; GENE ; GENE-EXPRESSION ; REPAIR ; DAMAGE ; LIPID-PEROXIDATION ; HUMAN-DISEASE ; HYPOMETHYLATION ; METHYLTRANSFERASE ; 3,N-4-ETHENOCYTOSINE ; LIVER DNA ; ADDUCT TYPES
    Abstract: Methylation of cytidine at dCpdG sequences regulates gene expression and is altered in many chronic inflammatory diseases. Inflammation generates lipid peroxidation (LPO) products which can react with deoxycytidine, deoxyadenosine, and deoxyguanosine in DNA to form pro-mutagenic exocyclic etheno-nucleoside residues. Since 5-methyl-2'-deoxycytidine (5mdC) residues exhibit increased nucleophilicity at N3, they should be even better targets for LPO products. We synthesized and characterized 3,N(4)-etheno-5-methyl-2'-deoxycytidine-3'-phosphate and showed that LPO products can indeed form the corresponding etheno-5mdC (epsilon 5mdC) lesion in DNA in vitro. Our newly developed (32)P-postlabeling method was subsequently used to detect epsilon 5mdC lesions in DNA from human white blood cells, lung, and liver at concentrations 4-10 times higher than that observed for etheno adducts on nonmethylated cytidine. Our new detection method can now be used to explore the hypothesis that this DNA lesion perturbs the DNA methylation status
    Type of Publication: Journal article published
    PubMed ID: 22148471
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  • 4
    Keywords: IN-VIVO ; LUNG-CANCER ; SYSTEM ; TISSUE ; DNA ; CARCINOGENESIS ; DIESEL EXHAUST ; AIR-POLLUTION ; RAT ; CONTAMINANT 3-NITROBENZANTHRONE ; RATS ; LINKAGE ; IDENTIFICATION ; genotoxicity ; HUMAN ACETYLTRANSFERASES ; METABOLIC-ACTIVATION ; POLLUTANT 3-NITROBENZANTHRONE ; Jun ; ADDUCTS ; rodent ; STANDARD ; V79 CELLS ; RE ; ADDUCT ; MUTAGEN 3-NITROBENZANTHRONE ; SULFOTRANSFERASES ; DNA ADDUCT
    Abstract: 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. 3-NBA forms DNA adducts in rodent tissues that arise principally through reduction to N-hydroxy-3-aminobenzanthrone (N-OHABA), esterification to its acetate or sulfate ester, and reaction of this activated ester with DNA. We detected 3-NBA-derived DNA adducts in rodent tissues by P-32-postlabeling and generated them chemically by acid-catalyzed reaction of N-OH-ABA with DNA, but their structural identification has not yet been reported. We have now prepared 3-NBA-derived adducts by reaction of a possible reactive metabolite, N-acetoxy-N-acetyl-3-aminobenzanthrone (N-Aco-N-Ac-ABA), with purine nucleosides and nucleotides, characterized them, and have shown that they are present in DNA treated with this 3-NBA derivative. Three of these adducts have been characterized as the C-C adduct N-acetyl-3-amino-2-(2'-deoxyguanosin-8-yl)benzanthrone, the C-N adduct N-acetyl-N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone, and an unusual 3-acetylaminobenzanthrone adduct of deoxyadenosine, which involves a double linkage between adenine and benzanthrone (N1 to C1, N6 to C11b), creating a five-membered imidazo type ring system. According to IUPAC fused ring conventions, we propose the following systematic name for this adduct: (9'-(2"-deoxyribofuranosyl))purino[6',1':2,3]imidazo[5,4-p]-(1,11b-dihyd ro-(N-acetyl-3-amino))benzanthrone. The X-phosphates of these novel adducts could be 5'-postlabeled using [gamma-P-32]ATP, although the efficiency of labeling was found to be low (less than 20%). However, none of these adducts could be detected in DNA from 3-NBA-treated rats by P-32-postlabeling. Two of these synthetic adducts were treated with alkali to generate nonacetylated adducts, and these were also shown by HPLC to differ from those adducts found in rat DNA. Therefore, a different approach to the synthesis of authentic standards is needed for the structural characterization of 3-NBA-derived DNA adducts formed in vivo
    Type of Publication: Journal article published
    PubMed ID: 15962941
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  • 5
    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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  • 6
    Abstract: Increased excretion of ethylated DNA bases has been reported in the urine of cigarette smokers. To study DNA ethylation in the target organs of smokers, an immunoenriched (32)P-postlabeling assay for O(4)-ethylthymidine (O(4)-etT) was developed. O(4)-etT-3'-monophosphate (O(4)-etT-3'P) was synthesized, purified, and characterized by LC-MS, ESI-MS, and NMR. DNA was enzymatically digested to 2'-deoxynucleoside-3'-monophosphate followed by immunoprecipitation of O(4)-etT-3'P using specific monoclonal antibodies. The immunoconjugate was washed by filtration, and O(4)-etT-3'P was recovered by ethanol treatment. The enriched O(4)-etT-3'P was labeled with [gamma-(32)P]ATP in the presence of T4-polynucleotide kinase at pH 6.8 to yield its 5'-labeled monophosphate and was subsequently resolved on RP-HPLC and detected with online detection of radioactivity. Adduct recovery was 〉80%, and the detection limit was approximately 500 amol. To further validate the method, O(4)-etT levels were determined in calf thymus DNA treated with N-ethyl-N-nitrosourea, and a dose-dependent formation of O(4)-etT was observed. Furthermore, O(4)-etT was found to be present in the cells obtained from the lower respiratory tract by sputum induction of two out of four smokers but not in three nonsmokers. O(4)-etT is a poorly repaired promutagenic DNA lesion; thus, it could be of potential use for biomonitoring smoking-related DNA damage. Our improved assay was found to be sufficiently sensitive and specific to detect O(4)-etT in surrogate cells from cigarette smoke exposed humans.
    Type of Publication: Journal article published
    PubMed ID: 11896692
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  • 7
    Keywords: CANCER ; EXPRESSION ; SYSTEM ; RISK ; MICE ; METABOLIC-ACTIVATION ; LINKED-IMMUNOSORBENT-ASSAY ; OXIDATIVE DNA-DAMAGE ; P-32-POSTLABELING ANALYSIS ; ANTICANCER DRUG ELLIPTICINE ; QUINONE OXIDOREDUCTASE ; NAD(P)H-QUINONE OXIDOREDUCTASE ; BENZENEDIAZONIUM ION ; ENVIRONMENTAL-POLLUTANT ; NON-AMINOAZO DYE
    Abstract: Sudan I (1-phenylazo-2-hydroxynaphthol) is a suspected human carcinogen causing tumors in the livers and urinary bladders of rats, mice, and rabbits. Here, we investigated for the first time the influence of Sudan I exposure on the expression of several biotransformation enzymes in the livers, kidneys, and lungs of rats concomitantly at the mRNA and protein levels and assayed their enzymatic activities. We also studied its effect on the formation of Sudan I-derived DNA adducts in vitro. Sudan I increased the total amounts of cytochrome P450 (P450) in all organs tested. Western blots using antibodies raised against various P450s, NADPH:P450 reductase, and NAD(P)H:quinone oxidoreductase 1 (NQO1) showed that the expression of P450 1A1 and NQO1 was induced in the liver, kidney, and lung of rats treated with Sudan I. The higher protein levels correlated with increased enzyme activities of P450 1A1/2 and NQO1. Furthermore, 9.9-, 5.9-, and 2.8-fold increases in the formation of Sudan I oxidative metabolites catalyzed by microsomes isolated from the liver, kidney, and lung, respectively, of rats treated with Sudan I were found. The relative amounts of P450 1A and NQO1 mRNA, measured by real-time polymerase chain reaction (RT-PCR) analysis, demonstrated that Sudan I induced the expression of P450 1A1 and NQO1 mRNA in the liver, kidney, and lung, and of P450 1A2 mRNA in kidney and lung. Finally, microsomes isolated from livers, kidneys, and lungs of Sudan I exposed rats more effectively catalyzed the formation of Sudan I-DNA adducts than microsomes from organs of control rats. This was attributable to the higher P450 1A1 expression. Because P450 1A1 is playing a major role in the bioactivation of Sudan I in rat and human systems, its induction by Sudan I may have a profound effect on cancer risk by this azo dye. In addition, the induction of P450 1A1/2 and NQO1 enzymes can influence individual human susceptibility to other environmental carcinogens and have an effect on cancer risk.
    Type of Publication: Journal article published
    PubMed ID: 23289503
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  • 8
  • 9
    Abstract: Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by (32)P-postlabeling was characterized as 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP.
    Type of Publication: Journal article published
    PubMed ID: 27404282
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  • 10
    Keywords: CANCER ; Germany ; human ; KINASE ; DNA adducts ; DNA ; CONTRAST ; SPECTROSCOPY ; FORM ; IDENTIFICATION ; IN-SITU ; ASSAY ; NMR ; BLADDER ; URINARY-BLADDER ; NUCLEOTIDES ; ATP ; Jun ; ADDUCTS ; P-32 POSTLABELING ANALYSIS ; MASSES ; STANDARD ; AGENT ; DNA-ADDUCTS ; thymus ; CARCINOGEN ; STANDARDS ; EXTRACTION ; 4-AMINOBIPHENYL-DNA ADDUCTS ; ALKYL SUBSTITUENTS ; CARCINOGEN-DNA ADDUCTS ; HUMAN UROEPITHELIAL CELLS ; MUTAGENIC AROMATIC-AMINES ; TOBACCO-SMOKE
    Abstract: The (32)p-postlabeling assay is an extremely sensitive technique for detecting carcinogen-DNA adducts. However, for the assignment of DNA adduct structures and the accurate determination of DNA adduct levels by (32)p-postlabeling, authentic adduct standards are needed. For Most P-32-postlabeling applications, such verified synthetic standard compounds are required in the form of their deoxynucleoside X-phosphates because they represent substrates for the polynucleotide kinase for transfer of [(32)p] phosphate from [gamma-P-32] ATP. Three N-(deoxyguanosin)4-aminobiphenyl X-phosphate adducts were prepared and fully characterized by H-1 NMR and mass spectroscopy to serve as standards for the 32P-postlabeling assay. Apart from the C8-and the N-2-deoxyguanosine X-phosphate adducts of the mutagenic human bladder carcinogen 4-aminobiphenyl (dG3'p-C8-4-ABP and dG3'p-N2-4-ABP), the C8-deoxyguanosine X-phosphate adduct of the nonmitagenic 4'-tert-butyl-4-aminobiphenyl (dG3'p-C8-4'tBu-4-ABP) was included in the study. Both C8-deoxyguanosine X-phosphate adducts were prepared by the in situ formation of deoxyguanosine X-phosphate and its subsequent reaction with the appropriate electrophilic amination agent (N-acetoxy compound). The N2-deoxyguanosine X-phosphate adduct was obtained by a modification of a previously described procedure for the synthesis of N2-deoxyguanosine adducts of aromatic amines. The three adduct standards were added at different concentrations to calf thymus DNA, and adduct recoveries were determined by the 32p-postlabeling assay under conditions routinely used in the standard methodology, enhancement by nuclease P1 digestion and enhancement by butanol extraction. The dG3'p-C8-4-ABP adduct was recovered irrespective of the concentration with approximately 30% in both the standard and the butanol extraction version of the assay. Both C8-deoxyguanosine X-phosphate adducts were sensitive to nuclease P1 digestion resulting in recoveries of only 1-3%. In contrast, the dG3'p-N2-4-ABP adduct was resistant to nuclease P1 digestion; however, recovery in all three versions was poor (1-2%) resulting in a detection limit of one adduct/10(6) nucleotides. These results demonstrate that the P-32-postlabeling assay underestimates the level of DNA adducts formed by 4-ABP and indicates that there is a need to determine the recovery for each adduct to be analyzed by the P-32-postlabeling technique
    Type of Publication: Journal article published
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