Urinary Metabolite Profiling Reveals CYP1A2-Mediated Metabolism of NSC686288 (Aminoflavone)

Abstract

NSC686288 [aminoflavone (AF)], a candidate chemotherapeutic agent, possesses a unique antiproliferative profile against tumor cells. Metabolic bioactivation of AF by drug-metabolizing enzymes, especially CYP1A monooxygenases, has been implicated as an underlying mechanism for its selective cytotoxicity in several cell culture-based studies. However, in vivo metabolism of AF has not been investigated in detail. In this study, the structural identities of 13 AF metabolites (12 of which are novel) in mouse urine or from microsomal incubations, including three monohydroxy-AFs, two dihydroxy-AFs and their sulfate and glucuronide conjugates, as well as one N-glucuronide, were determined by accurate mass measurements and liquid chromatography-tandem mass spectrometry fragmentation patterns, and a comprehensive map of the AF metabolic pathways was constructed. Significant differences between wild-type and Cyp1a2-null mice, within the relative composition of urinary metabolites of AF, demonstrated that CYP1A2-mediated regioselective oxidation was a major contributor to the metabolism of AF. Comparisons between wild-type and CYP1A2-humanized mice further revealed interspecies differences in CYP1A2-mediated catalytic activity. Incubation of AF with liver microsomes from all three mouse lines and with pooled human liver microsomes confirmed the observations from urinary metabolite profiling. Results from enzyme kinetic analysis further indicated that in addition to CYP1A P450s, CYP2C P450s may also play some role in the metabolism of AF.

Aminoflavone induces oxidative DNA damage and reactive oxidative species‐mediated apoptosis in breast cancer cells

“…Previous reports indicate that CYP1A2 metabolizes AF into a number of different species that have been characterized including a hydroxylated metabolite. 11 A recent study suggests that flavonoid small molecules with hydroxylation in the 3 position have a greater propensity to demonstrate prooxidant characteristics than those lacking this substitution. 40 We postulate that CYP1A2 promotes the metabolism of AF to a 3-hydroxylated derivative which may contribute to the prooxidant characteristics of AF as modeled in Figure 5d.…”

“…8 Previous studies have characterized and identified covalent drug adducts which indicates that AF metabolite(s) alkylate DNA. 8,11 Though AF may alkylate DNA, it likely possesses a mechanism of anticancer activity distinct from most alkylating agents also known to induce DPC formation. Studies are underway to identify the proteins involved in DPC formation following AF treatment which could shed additional light into its unique mechanism of action.…”

“…10,11 The ability of AF to induce its own metabolism reveals a unique aspect of its mechanism of anticancer action, a feature not found among clinically available anticancer agents. Sensitive cells possess constitutively high levels of sulfotransferases which can convert AF into active species and depend upon AhR signaling pathway activation resulting in CYP1A1 or CYP1A2 induction in response to the apoptotic and DNA damaging effects of AF.…”

LC-MS-Based Metabolomics in Drug Metabolism

“…Thirteen aminoflavone metabolites appear prominently in the loadings plot as the ions that greatly contribute to the separation of aminoflavone treatment from vehicle treatment in the first PC ( Fig. 2C; Chen et al, 2006).…”

Characterization of Differential Cocaine Metabolism in Mouse and Rat through Metabolomics-Guided Metabolite Profiling

“…Pooled human liver microsomes were purchased from Puracyp (Carlsbad, CA). Mice and rat liver microsomes were prepared from male C57BL/6 mice and male CD rats, respectively (Chen et al, 2006). The microsomal protein concentration was determined by bicinchoninic acid protein assay.…”