title: Characterization of an etoposide-glutathione conjugate derived from metabolic activation by human cytochrome p450.
abstract: Etoposide (VP-16), a DNA topoisomerase II poison widely used as an antineoplastic agent is also known to cause leukemia. One of its major metabolic pathways involves O-demethylation to etoposide catechol (etoposide-OH) by cytochrome P450 3A4 (CYP3A4). The catechol metabolite can undergo sequential one- and two-electron oxidations to form etoposide semi-quinone (etoposide-SQ) and etoposide quinone (etoposide-Q), respectively, which have both been implicated as cytotoxic metabolites. However, etoposide-Q is known to react with glutathione (GSH), which can protect DNA from oxidative damage by this reactive metabolite. In this study, etoposide-Q was reacted with GSH and the two etoposide-GSH conjugates were characterized. The major conjugate was etoposide-OH-6'-SG and the minor product was etoposide-OH-2'-SG. Etoposide-OH-6'-SG, which arose from Michael addition of GSH to etoposide-Q, was characterized by mass spectrometry and 2-D NMR. It was identified as the sole product from in vitro metabolism experiments using recombinant human CYP3A4 or liver microsomes incubated with etoposide in the presence of GSH. Etoposide-OH-6'-SG was also detected from incubations of etoposide-OH and GSH alone. Therefore, the presence of etoposide-OH, which can be formed from etoposide metabolism by CYP3A4, is essential for formation of the GSH conjugate. The oxidation of etoposide-OH to a quinone intermediate is likely the precursor in the formation of etoposide-OH-6'-SG.
authors.affiliation: Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, 854 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160, USA.
authors.forename: Naiyu
authors.initials: N
authors.lastname: Zheng
authors.forename: Shaokun
authors.initials: S
authors.lastname: Pang
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Oe
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Felix
authors.forename: Suzanne
authors.initials: S
authors.lastname: Wehrli
authors.forename: Ian A
authors.initials: IA
authors.lastname: Blair
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism; D002853:Chromatography, Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005047:Etoposide / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D005047:Etoposide; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D005978:Glutathione