Formation and Metabolism of 15-Deoxy-{Delta}12,14-Prostaglandin J2 in Vivo

Abstract

PHARMACOLOGY

FORMATION AND METABOLISM OF 15-DEOXY-{Delta}12,14-PROSTAGLANDIN J2 IN VIVO

KLARISSA D. HARDY

Dissertation under the direction of Professor L. Jackson Roberts, II

15-Deoxy-{Delta}12,14-prostaglandin J2 (15-d-PGJ2) is a reactive cyclopentenone PG generated from the dehydration of cyclooxygenase (COX)-derived PGD2, and this compound has emerged as a putative mediator of numerous biological effects. The cyclopentenone ring of 15-d-PGJ2 contains an electrophilic alpha,beta-unsaturated carbonyl moiety which can readily undergo nucleophilic addition reactions (Michael addition) with cellular thiols, such as glutathione and cysteine residues of proteins. Due to its reactivity, 15-d-PGJ2 can modulate protein function and exerts potent anti-inflammatory and pro-apoptotic activity in various cell types. The biological effects of 15-d-PGJ2 are mediated in part through activation of the nuclear receptor peroxisome proliferator-activated receptor-gamma as well as through direct interaction with other intracellular protein targets, such as transcription factors and signaling molecules. However, evidence for the biosynthesis of 15-d-PGJ2 in vivo is lacking. The goal of this dissertation research is to determine the extent to which 15-d-PGJ2 is formed in vivo and examine the mechanisms that contribute to its formation. We hypothesized that 15-d-PGJ2 may be generated in vivo via the COX and free radical-catalyzed pathways. Through the studies described herein, we have found that 15-d-PGJ2 and a series of 15-d-PGJ2-like compounds (deoxy-J2-IsoPs) are generated in vivo via free radical-initiated lipid peroxidation after induction of oxidative stress, independent of COX. The formation of these reactive compounds has potential relevance to the pathobiology of diseases associated with oxidative stress. This finding provided the impetus for us to examine the systemic generation of 15-d-PGJ2 and deoxy-J2-IsoPs in humans. Due to the reactivity of these molecules, we hypothesized that they undergo rapid adduction to protein and peptide thiols. Thus, we determined the metabolic fate of 15-d-PGJ2 in primary human cells and in vivo in the rat. From these studies, we identified several metabolites of 15-d-PGJ2 in primary human hepatocytes. We also identified the major urinary metabolite of 15-d-PGJ2 in the rat, and our results suggest that this metabolite is formed in humans. Importantly, the identification of this metabolite provides a potential biomarker to quantify the production of 15-d-PGJ2 and related compounds in vivo in humans under various physiological and pathophysiological conditions.