title: The cyclopentenone product of lipid peroxidation, 15-A2t-isoprostane, is efficiently metabolized by HepG2 cells via conjugation with glutathione.
abstract: Cyclopentenone isoprostanes (IsoPs), A(2)/J(2)-IsoPs, are one class of IsoPs formed via the free radical-initiated peroxidation of arachidonic acid. These compounds, which are structurally similar to cyclooxygenase-derived PGA(2) and PGJ(2), contain highly reactive alpha,beta-unsaturated carbonyl moieties. A(2)/J(2)-IsoPs are generated in vivo in humans esterified in glycerophospholipids. Unlike other classes of IsoPs, however, cyclopentenone IsoPs cannot be detected in the free form; we postulated that this might be due to their rapid adduction to various thiol-containing biomolecules via Michael addition. Recently, we reported that the A-ring IsoP, 15-A(2t)-IsoP, is efficiently conjugated with glutathione in vitro by certain human and rat glutathione transferases (GSTs), with the isozyme GSTA4-4 displaying the highest activity. Herein, we examined the metabolic disposition of 15-A(2t)-IsoP in HepG2 cells. We report that 15-A(2t)-IsoP is primarily metabolized by these cells via conjugation to glutathione. Within 6 h, approximately 60% of 15-A(2t)-IsoP added to HepG2 cells was present in the form of a water soluble conjugate(s). Structural characterization of the adduct(s) by liquid chromatography-tandem mass spectrometry revealed four major conjugates. These include the intact 15-A(2t)-IsoP-GSH conjugate, the GSH conjugate in which the carbonyl at C-9 of 15-A(2t)-IsoP is reduced, and the corresponding cysteine conjugates. These studies thus show that the primary pathway of metabolic disposition of endogenously derived cyclopentenone IsoPs occurs via conjugation with thiols.
authors.affiliation: Department of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Ginger L
authors.initials: GL
authors.lastname: Milne
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Zanoni
authors.forename: Alessio
authors.initials: A
authors.lastname: Porta
authors.forename: Soumya
authors.initials: S
authors.lastname: Sasi
authors.forename: Giovanni
authors.initials: G
authors.lastname: Vidari
authors.forename: Erik S
authors.initials: ES
authors.lastname: Musiek
authors.forename: Michael L
authors.initials: ML
authors.lastname: Freeman
authors.forename: Jason D
authors.initials: JD
authors.lastname: Morrow
mesh_terms: D003517:Cyclopentanes / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015227:Lipid Peroxidation; D011454:Prostaglandins A / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014316:Tritium; D014407:Tumor Cells, Cultured
chemical_list: C117466:8-isoprostaglandin A2; D003517:Cyclopentanes; D011454:Prostaglandins A; D014316:Tritium; D005978:Glutathione; D003545:Cysteine; C013905:cyclopentenone