Cancer Research UK Molecular Pharmacology Unit

Dundee, United Kingdom

Cancer Research UK Molecular Pharmacology Unit

Dundee, United Kingdom
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Potega A.,Technical University of Gdansk | Dabrowska E.,Technical University of Gdansk | Niemira M.,Technical University of Gdansk | Kot-Wasik A.,Technical University of Gdansk | And 4 more authors.
Drug Metabolism and Disposition | Year: 2011

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor agent that is also active against autoimmune diseases. The intention of the present studies was to elucidate the role of selected liver enzymes in metabolism of C-1311 and the less active 8-methyl derivative, 5-diethylaminoethylamino-8-methoxyimidazoacridinone (C-1330). Compounds were incubated with rat liver microsomal fraction, with a set of 16 human liver protein samples, and with human recombinant isoenzymes of cytochrome P450, flavin monooxygenases (FMO), and UDP-glucuronosyltransferase (UGT). Our results showed that C-1311 and C-1330 were metabolized with human liver microsomal enzymes but not with any tested human recombinant cytochromes P450 (P450s). Two of these, CYP1A2 and CYP3A4, were inhibited by both compounds. In addition, results of C-1311 elimination from hepatic reductase-null mice, in which liver NADPH-P450 oxidoreductase has been deleted indicated that liver P450s were slightly engaged in drug transformation. In contrast, both compounds were good substrates for human recombinant FMO1 and FMO3 but not for FMO5. The product of FMO metabolism, PFMO, which is identified as anN-oxide derivative, was identical to P3 R of liver microsomes. P3 R was observed even in the presence of the P450 inhibitor, 1-aminobenzotriazole, and it disappeared after heating. Therefore, FMO enzymes could be responsible for microsomal metabolism to P3 R = P FMO. Glucuronidation on the 8-hydroxyl group of C-1311 was observed with liver microsomes supported by UDP-glucuronic acid and with recombinant UGT1A1, but it was not the case with UGT2B7. Summing up, we showed that, whereas liver P450 isoenzymes were involved in the metabolism of C-1311 to a limited extent, FMO plays a significant role in the microsomal transformations of this compound, which is also a specific substrate of UGT1A1. Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics.


Singh R.,University of Leicester | Arlt V.M.,Institute of Cancer Research | Henderson C.J.,Cancer Research UK Molecular Pharmacology Unit | Phillips D.H.,Institute of Cancer Research | And 2 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2010

The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed by the grilled cooking of certain foods such as meats, poultry and fish. PhIP has been shown to induce tumours in the colon, prostate and mammary glands of rats and is regarded as a potential human dietary carcinogen. PhIP is metabolically activated via cytochrome P450 mediated oxidation to an N-hydroxylamino-PhIP intermediate that is subsequently converted to an ester by N-acetyltransferases or sulfotransferases and undergoes heterolytic cleavage to produce a PhIP-nitrenium ion, which reacts with DNA to form the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) adduct. Thus far, the detection and quantification of PhIP-DNA adducts has relied to a large extent on 32P-postlabelling methodologies. In order to expand the array of available techniques for the detection and improved quantification of PhIP-C8-dG adducts in DNA we have developed an online column-switching liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) method incorporating an isotopically [13C10]-labelled PhIP-C8-dG internal standard for the analysis of DNA enzymatically hydrolysed to 2'-deoxynucleosides. A dose-dependent increase was observed for PhIP-C8-dG adducts when salmon testis DNA was reacted with N-acetoxy-PhIP. Analysis of DNA samples isolated from colon tissue of mice treated by oral gavage daily for 5 days with 50mg/kg body weight of PhIP resulted in the detection of an average level of 14.8±3.7 PhIP-C8-dG adducts per 106 2'-deoxynucleosides. The method required 50μg of hydrolysed animal DNA on column and the limit of detection for PhIP-C8-dG was 2.5fmol (1.5 PhIP-C8-dG adducts per 108 2'-deoxynucleosides). In summary, the LC-ESI-MS/MS SRM method provides for the rapid automation of the sample clean up and a reduction in matrix components that would otherwise interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP. © 2010 Elsevier B.V.

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