The cyclopentenone product of lipid peroxidation, 15-A2t-isoprostane, is efficiently metabolized by HepG2 cells via conjugation with glutathione.
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.
Milne
Ginger L
GL
Department of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Zanoni
Giuseppe
G
Porta
Alessio
A
Sasi
Soumya
S
Vidari
Giovanni
G
Musiek
Erik S
ES
Freeman
Michael L
ML
Morrow
Jason D
JD
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
C117466:8-isoprostaglandin A2; D003517:Cyclopentanes; D011454:Prostaglandins A; D014316:Tritium; D005978:Glutathione; D003545:Cysteine; C013905:cyclopentenone