UNLABELLED: Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H: quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using (32)P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast, the formation of AAI-DNA adducts was catalyzed by CYP1B1 with the A133S mutation. Our experimental model confirms the importance of the hydroxyl group possessing amino acids in the active center of CYP1A1 and 1A2 for AAI nitroreduction.
- MeSH
- adukty DNA metabolismus MeSH
- aromatické hydroxylasy genetika metabolismus MeSH
- cytochrom P-450 CYP1A1 MeSH
- cytochrom P-450 CYP1A2 MeSH
- cytochrom P450 CYP1B1 MeSH
- katalytická doména genetika MeSH
- katalýza MeSH
- kyseliny aristolochové metabolismus MeSH
- lidé MeSH
- mutace * MeSH
- mutageneze cílená MeSH
- oxidace-redukce MeSH
- rekombinantní proteiny MeSH
- substrátová specifita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Cytochromes P450 (CYPs) are heme enzymes oxygenating a broad range of substrates. Their activity is dependent on the presence of a suitable electron donor (eukaryotic NADPH:CYP oxidoreductase or cytochrome b5). The Escherichia naturally contain no CYPs and no NADPH:CYP oxidoreductase, however it was reported that some CYPs heterologously expressed in E. coli may exist in the ferrous form. A small bacterial flavoprotein, flavodoxin is considered to be responsible for reduction some of these CYPs. METHODS: The reduction state of several human CYPs expressed in the intact living E. coli cells was examined. In addition, molecular dynamics and steered molecular dynamics simulations were performed to predict and compare affinity of flavodoxin toward selected CYPs. RESULTS: We determined the reduction state of five human CYPs heterologously expressed in E. coli. The computationally predicted stabilities of CYP-flavodoxin complexes correlate with the percentage of reduced CYPs in bacterial cells. The mean electron transfer distance within optimized complexes was also related to the percentage of reduced CYPs. CONCLUSION: Depending on the resting state, the CYPs heterologously expressed in E. coli could be divided into two groups; CYP2C8, 2C9, 3A4 are in E. coli present mainly in the oxidized form; while CYP1A1, 1A2, 2A6, 2A13, 2B6, 2D6 are found predominantly in the reduced form. We found a significant correlation between the stability of CYP-flavodoxin complexes and the percentage of reduced CYPs in bacteria. Hence, the naturally expressed flavodoxin is probably responsible for reduction of a larger group of human CYPs in bacterial cells.
- MeSH
- aromatické hydroxylasy metabolismus MeSH
- cytochrom P-450 CYP1A1 metabolismus MeSH
- cytochrom P-450 CYP1A2 metabolismus MeSH
- cytochrom P-450 CYP2D6 metabolismus MeSH
- cytochrom P-450 CYP3A metabolismus MeSH
- cytochrom P450 CYP2A6 metabolismus MeSH
- cytochrom P450 CYP2B6 metabolismus MeSH
- cytochrom P450 CYP2C8 metabolismus MeSH
- cytochrom P450 CYP2C9 metabolismus MeSH
- Escherichia coli MeSH
- flavodoxin metabolismus MeSH
- geneticky modifikované organismy MeSH
- lidé MeSH
- NADPH-cytochrom c-reduktasa metabolismus MeSH
- oxidace-redukce MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- železité sloučeniny metabolismus MeSH
- železnaté sloučeniny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- adukty DNA účinky léků metabolismus MeSH
- barvicí látky metabolismus MeSH
- cytochrom P-450 CYP1A1 genetika metabolismus MeSH
- cytosol účinky léků enzymologie MeSH
- játra účinky léků enzymologie MeSH
- karcinogeny metabolismus MeSH
- krysa rodu rattus MeSH
- ledviny účinky léků enzymologie MeSH
- messenger RNA genetika MeSH
- mikrozomy účinky léků enzymologie MeSH
- NAD(P)H dehydrogenasa (chinon) genetika metabolismus MeSH
- naftoly metabolismus MeSH
- plíce účinky léků enzymologie MeSH
- potkani Wistar MeSH
- upregulace účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
