title: 2'-Deoxyguanosine as a surrogate trapping agent for DNA reactive drug metabolites.
abstract: Drug metabolism can result in the production of highly reactive metabolites that may form adducts with cellular macromolecules, and thus initiate adverse drug reactions, cause toxicity, and even require the withdrawal of drug from the market. In this study, a 2'-deoxyguanosine (dG)-based chemical trapping test system was developed for use as a fast screening tool for DNA adducting metabolites of new drug candidates. Reactive metabolites were generated from parent compounds in in vitro incubations with phenobarbital-induced mouse liver microsomes, human liver microsomes and different recombinant human CYP enzymes in the presence of dG. The formed dG-adducts were separated, characterized and their stability was studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was evaluated with six test compounds, aflatoxin B1, estrone, clozapine, tolcapone, ticlopidine and imipramine. Estrone and aflatoxin B1 formed dG adducts with phenobarbital-induced mouse liver microsomes, human liver microsomes and human recombinant CYP enzymes. Adduct formation was also observed with tolcapone when phenobarbital-induced mouse liver microsomes were used as the enzyme source. The stability of each formed adduct was independent of the different enzyme sources. No dG-adducts were identified with ticlopidine, clozapine and imipramine. Compared to other classical DNA reactivity tests, e.g. Ames test, the present surrogate endpoint, the dG adduct, is faster, enables the characterization of the formed compounds, and also permits the investigation of more unstable adducts.
authors: School of Pharmacy, University of Eastern Finland, Kuopio Campus, PO Box 1627, FI70211 Kuopio, Finland. Merja.Hakkinen@uef.fi
authors: Merja R
authors: MR
authors: Häkkinen
authors: Jaana E
authors: JE
authors: Laine
authors: Risto O
authors: RO
authors: Juvonen
authors: Seppo
authors: S
authors: Auriola
authors: Jukka
authors: J
authors: Häyrinen
authors: Markku
authors: M
authors: Pasanen
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D018736:DNA Adducts / Q000032:analysis*; D003849:Deoxyguanosine / Q000378:metabolism*; D005260:Female; D006801:Humans; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008811:Mice, Inbred DBA; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D018736:DNA Adducts; D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System; D003849:Deoxyguanosine