Ellipticine and benzo(a)pyrene increase their own metabolic activation via modulation of expression and enzymatic activity of cytochromes P450 1A1 and 1A2

Two compounds known to covalently bind to DNA after their activation with cytochromes P450 (CYPs), carcinogenic benzo(a)pyrene (BaP) and an antineoplastic agent ellipticine, were investigated for their potential to induce CYP and NADPH:CYP reductase (POR) enzymes in rodent livers, the main target organ for DNA adduct formation. Two animal models were used in the study: (i) rats as animals mimicking the fate of ellipticine in humans and (ii) mice, especially wild-type (WT) and hepatic POR null (HRN™) mouse lines. Ellipticine and BaP induce expression of CYP1A enzymes in livers of experimental models, which leads to increase in their enzymatic activity. In addition, both compounds are capable of generating DNA adducts, predominantly in livers of studied organisms. As determined by 32P postlabelling analysis, levels of ellipticine-derived DNA adducts formed in vivo in the livers of HRN™ mice were reduced (by up to 65%) relative to levels in WT mice, indicating that POR mediated CYP enzyme activity is important for the activation of ellipticine. In contrast to these results, 6.4 fold higher DNA binding of BaP was observed in the livers of HRN™ mice than in WT mice. This finding suggests a detoxication role of CYP1A in BaP metabolism in vivo. In in vitro experiments, DNA adduct formation in calf thymus DNA was up to 25 fold higher in incubations of ellipticine or BaP with microsomes from pretreated animals than with controls. This stimulation effect was attributed to induction of CYP1A1/2 enzymes, which are responsible for oxidative activation of both compounds to the metabolites generating major DNA adducts in vitro. Taken together, these results demonstrate that by inducing CYP1A1/2, ellipticine and BaP modulate their own enzymatic metabolic activation and detoxication, thereby modulating their either pharmacological (ellipticine) and/or genotoxic potential (both compounds)

Analysis of benzo[a]pyrene metabolites formed by rat hepatic microsomes using high pressure liquid chromatography: optimization of the method

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

Study of the molecular mechanism of anticancer drug ellipticine

Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

Study of the molecular mechanism of anticancer drug ellipticine

Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

Metabolism carcinogens and drugs by the system of monooxygenases

Ellipticine, an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activities. Ellipticine is a pro-drug, whose pharmacological and genotoxic effects depend on activation by cytochromes P450 (CYP) and peroxidases (Px) to a reactive species generating DNA adducts. To elucidate contribution of CYPs (and which of them) and Px to ellipticine activation, we used rat and mouse models, mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in the liver (HRNTM ) and a control mouse line (WT), rats treated with ellipticine, and microsomal systems isolated from the liver of mouse lines and from the liver, kidney and lung of rats. The purified enzymes, CYP1A1 and 3A4, reconstituted with NADPH:CYP reductase were also used. The effect of cytochrome b5, a facultative component of the mixed function monooxygenase system, on ellipticine oxidation by CYP1A1 and 3A4 was also investigated. Carcinogenic benzo(a)pyrene (BaP), known to covalently bind to DNA after its activation with CYPs, was investigated for its potential to generate DNA adducts and to induce CYP and NADPH:CYP reductase enzymes in mouse livers. We investigated an influence of each of components of the mixed function oxidases (MFO) system on metabolism of BaP. CYP1A1 is widely accepted to be the..

Study of the molecular mechanism of anticancer drug ellipticine

Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

NADPH- and NADH-dependent metabolism of and DNA adduct formation by benzo[a]pyrene catalyzed with rat hepatic microsomes and cytochrome P450 1A1

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: [Image: see text

Induction of cytochromes P450 1A1 and 1A2 suppresses formation of DNA adducts by carcinogenic aristolochic acid I in rats in vivo

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