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  • CHINESE HERBS NEPHROPATHY  (2)
  • 1
    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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  • 2
    Keywords: CANCER ; IN-VITRO ; INHIBITOR ; human ; liver ; SITE ; ENZYMES ; SAMPLE ; SAMPLES ; METABOLISM ; TISSUE ; TIME ; ACTIVATION ; DNA ; kidney ; renal ; DNA ADDUCT FORMATION ; metabolic activation ; RAT ; TISSUES ; SUSCEPTIBILITY ; ACID ; RAT-LIVER ; PATTERNS ; ASSAY ; MODULATION ; POLYCYCLIC AROMATIC-HYDROCARBONS ; HUMAN LIVER ; rat liver ; ORIENTATION ; HUMAN-LIVER ; aristolochic acid ; aldehyde ; BALKAN ENDEMIC NEPHROPATHY ; CHINESE HERBS NEPHROPATHY ; DT-DIAPHORASE ; P-32 POSTLABELING ANALYSIS ; QUINONE REDUCTASE ; RAT-LIVER CYTOSOL ; XANTHINE-OXIDASE
    Abstract: Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA metabolism is important in the assessment of an individual's susceptibility to this carcinogen. Using the P-32-postlabeling assay we examined the ability of enzymes of cytosolic samples from 10 different human livers and from one human kidney to activate the major component of the plant extract AA, 8-methoxy- 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI), to metabolites forming adducts in DNA. Cytosolic fractions of both organs generated AAI-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, indicating a possible demethoxylation reaction of AAI, were identified as AA-DNA adducts formed from AAI by all human hepatic and renal cytosols. To define the role of human cytosolic reductases in the activation of AAI, we investigated the modulation of AAI-DNA adduct formation by cofactors or selective inhibitors of the NAD(P)H:quinone oxidoreductase (NQO1), xanthine oxidase (XO) and aldehyde oxidase. We also determined whether the activities of NQO1 and XO in different human hepatic cytosolic samples correlated with the levels of AAI-DNA adducts formed by the same cytosolic samples. Based on these studies, we attribute most of the activation of AA in human cytosols to NQO1, although a role of cytosolic XO cannot be ruled out. With purified NQO1 from rat liver and kidney and XO from buttermilk, the major role of NQO1 in the formation of AAI-DNA adducts was confirmed. The orientation of AAI in the active site of human NQO1 was predicted from molecular modeling based on published X-ray structures. The results demonstrate for the first time the potential of human NQO1 to activate AAI by nitroreduction
    Type of Publication: Journal article published
    PubMed ID: 12869422
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