UNLABELLED: Aristolochic acid I (AAI) is a natural plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. One of the most efficient enzymes reductively activating AAI to species forming AAI-DNA adducts is cytosolic NAD(P)H: quinone oxidoreductase 1. AAI is also either reductively activated or oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by microsomal cytochrome P450 (CYP) 1A1 and 1A2. Here, we investigated which of these two opposing CYP1A1/2-catalyzed reactions prevails in AAI metabolism in vivo. The formation of AAI-DNA adducts was analyzed in liver, kidney and lung of rats treated with AAI, Sudan I, a potent inducer of CYP1A1/2, or AAI after pretreatment with Sudan I. Compared to rats treated with AAI alone, levels of AAI-DNA adducts determined by the (32)P-postlabeling method were lower in liver, kidney and lung of rats treated with AAI after Sudan I. The induction of CYP1A1/2 by Sudan I increased AAI detoxification to its O-demethylated metabolite AAIa, thereby reducing the actual amount of AAI available for reductive activation. This subsequently resulted in lower AAI-DNA adduct levels in the rat in vivo. Our results demonstrate that CYP1A1/2-mediated oxidative detoxification of AAI is the predominant role of these enzymes in rats in vivo, thereby suppressing levels of AAI-DNA adducts.

• Klíčová slova
• Aristolochic acid I, Cytochromes P450 1A1 and 1A2, DNA adducts, Oxidative detoxification, Reductive activation,
• MeSH
• adukty DNA antagonisté a inhibitory   biosyntéza   MeSH
• cytochrom P-450 CYP1A1 biosyntéza   MeSH
• cytochrom P-450 CYP1A2 biosyntéza   MeSH
• enzymová indukce účinky léků   fyziologie   MeSH
• karcinogeny toxicita   MeSH
• krysa rodu Rattus MeSH
• kyseliny aristolochové toxicita   MeSH
• potkani Wistar MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• aristolochic acid I MeSH Prohlížeč
• CYP1A1 protein, human MeSH Prohlížeč
• CYP1A2 protein, human MeSH Prohlížeč
• cytochrom P-450 CYP1A1 MeSH
• cytochrom P-450 CYP1A2 MeSH
• karcinogeny MeSH
• kyseliny aristolochové MeSH

ABSTRACT: The microsomal protein cytochrome b5 , which is located in the membrane of the endoplasmic reticulum, has been shown to modulate many reactions catalyzed by cytochrome P450 (CYP) enzymes. We investigated the influence of exposure to the anticancer drug ellipticine and to two environmental carcinogens, benzo[a]pyrene (BaP) and 1-phenylazo-2-naphthol (Sudan I), on the expression of cytochrome b5 in livers of rats, both at the mRNA and protein levels. We also studied the effects of these compounds on their own metabolism and the formation of DNA adducts generated by their activation metabolite(s) in vitro. The relative amounts of cytochrome b5 mRNA, measured by real-time polymerase chain reaction analysis, were induced by the test compounds up to 11.7-fold in rat livers. Western blotting using antibodies raised against cytochrome b5 showed that protein expression was induced by up to sevenfold in livers of treated rats. Microsomes isolated from livers of exposed rats catalyzed the oxidation of ellipticine, BaP, and Sudan I and the formation of DNA adducts generated by their reactive metabolite(s) more effectively than hepatic microsomes isolated from control rats. All test compounds are known to induce CYP1A1. This induction is one of the reasons responsible for increased oxidation of these xenobiotics by microsomes. However, induction of cytochrome b5 can also contribute to their enhanced metabolism.

• Klíčová slova
• DNA, Drug research, Enzymes, High pressure liquid chromatography,
• Publikační typ
• časopisecké články MeSH

Aristolochic acid I (AAI) is a plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is detoxified by cytochrome P450 (CYP)-mediated O-demethylation to 8-hydroxyaristolochic acid I (aristolochic acid Ia, AAIa). We previously investigated the efficiencies of human and rat CYPs in the presence of two other components of the mixed-functions-oxidase system, NADPH:CYP oxidoreductase and cytochrome b₅, to oxidize AAI. Human and rat CYP1A are the major enzymes oxidizing AAI. Other CYPs such as CYP2C, 3A4, 2D6, 2E1, and 1B1, also form AAIa, but with much lower efficiency than CYP1A. Based on velocities of AAIa formation by examined CYPs and their expression levels in human and rat livers, here we determined the contributions of individual CYPs to AAI oxidation in these organs. Human CYP1A2 followed by CYP2C9, 3A4 and 1A1 were the major enzymes contributing to AAI oxidation in human liver, while CYP2C and 1A were most important in rat liver. We employed flexible in silico docking methods to explain the differences in AAI oxidation in the liver by human CYP1A1, 1A2, 2C9, and 3A4, the enzymes that all O-demethylate AAI, but with different effectiveness. We found that the binding orientations of the methoxy group of AAI in binding centers of the CYP enzymes and the energies of AAI binding to the CYP active sites dictate the efficiency of AAI oxidation. Our results indicate that utilization of experimental and theoretical methods is an appropriate study design to examine the CYP-catalyzed reaction mechanisms of AAI oxidation and contributions of human hepatic CYPs to this metabolism.

• Klíčová slova
• contribution of cytochromes P450 in detoxification of aristolochic acid I in human and rat livers, cytochrome P450-mediated detoxification of aristolochic acid I, molecular modeling, plant nephrotoxin and carcinogen aristolochic acid I,
• MeSH
• inhibitory cytochromu P450 farmakologie   MeSH
• jaterní mikrozomy účinky léků   metabolismus   MeSH
• játra účinky léků   metabolismus   MeSH
• katalytická doména MeSH
• katalýza MeSH
• krysa rodu Rattus MeSH
• kyseliny aristolochové škodlivé účinky   chemie   metabolismus   MeSH
• lidé MeSH
• metabolická aktivace MeSH
• metabolická inaktivace MeSH
• metylace účinky léků   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• nemoci ledvin etiologie   metabolismus   MeSH
• oxidace-redukce účinky léků   MeSH
• systém (enzymů) cytochromů P-450 chemie   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aristolochic acid I MeSH Prohlížeč
• inhibitory cytochromu P450 MeSH
• kyseliny aristolochové MeSH
• systém (enzymů) cytochromů P-450 MeSH

Sudan I [1-(phenylazo)-2-hydroxynaphthalene, C.I. Solvent Yellow 14, CAS No: 842-07-9] is used as the compound employed in chemical industry and to color materials such as hydrocarbon solvents, oils, fats, waxes, plastics, printing inks, shoe and floor polishes and gasoline. Such a wide used could result in a considerable human exposure. Sudan I is known to cause developments of tumors in the liver or urinary bladder in rats, mice, and rabbits, and is considered a possible weak human carcinogen and mutagen. This carcinogen is also a potent contact allergen and sensitizer. Here, we compare the data concerning the Sudan I oxidative metabolism catalyzed by cytochrome P450 (CYP) and peroxidase enzymes, which has been investigated in our laboratory during the last two decades. These two types of enzymes are responsible both for Sudan I detoxication and activation. Among the Sudan I metabolites, C-hydroxylated derivatives and a dimer of Sudan I are suggested to be the detoxication metabolites formed by CYPs and peroxidases, respectively. Metabolic activation of Sudan I by both types of enzymes leads to formation of reactive species (the benzenediazonium ion by CYP and Sudan I radicals by peroxidase) that bind to DNA and RNA, generating covalent adducts in vitro and in vivo. Whereas the structure of the major adduct formed by the benzenediazonium ion in DNA has already been identified to be the 8-(phenylazo)guanine adduct, the structures of adducts formed by peroxidase, have not been characterized as yet. Biological significance of the DNA adducts of Sudan I activated with CYP and peroxidase enzymes and further aims of investigations in this field are discussed in this study.

• Klíčová slova
• Sudan I, carcinogenic azo dye, cytochrome P450, oxidative activation, peroxidase,
• Publikační typ
• časopisecké články MeSH