Olivier Boutaud

Determinants of the cellular specificity of acetaminophen as an inhibitor of prostaglandin H2 synthases

Acetaminophen has antipyretic and analgesic properties yet differs from the nonsteroidal antiinflammatory drugs and inhibitors of prostaglandin H synthase (PGHS)-2 by exhibiting little effect on platelets or inflammation. We find parallel selectivity at a cellular level; acetaminophen inhibits PGHS activity with an IC50 of 4.3 μM in interleukin (IL)-1α-stimulated human umbilical vein endothelial cells, in contrast with an IC50 of 1,870 μM for the platelet, with 2 μM arachidonic acid as substrate. This difference is not caused by isoform selectivity, because acetaminophen inhibits purified ovine PGHS-1 and murine recombinant PGHS-2 equally. We explored the hypothesis that this difference in cellular responsiveness results from antagonism of the reductant action of acetaminophen on the PGHSs by cellular peroxides. Increasing the peroxide product of the PGHS-cyclooxygenase, prostaglandin G2 (PGG2), by elevating the concentration of either enzyme or substrate reverses the inhibitory action of acetaminophen, as does the addition of PGG2 itself. 12-Hydroperoxyeicosatetraenoic acid (0.3 μM), a major product of the platelet, completely reverses the action of acetaminophen on PGHS-1. Inhibition of PGHS activity by acetaminophen in human umbilical vein endothelial cells is abrogated by t-butyl hydroperoxide. Together these findings support the hypothesis that the clinical action of acetaminophen is mediated by inhibition of PGHS activity, and that hydroperoxide concentration contributes to its cellular selectivity.

New insights into the mechanism of action of acetaminophen: Its clinical pharmacologic characteristics reflect its inhibition of the two prostaglandin H2 synthases.

a t s o o t Acetaminophen (INN, paracetamol) possesses highly elective analgesic and antipyretic effects that result rom its inhibitory actions on the synthesis of prostalandins (PGs). PGs are lipid mediators derived from rachidonic acid that play central roles in the pathogensis of inflammation, fever, and pain. However, acetminophen differs from the majority of nonsteroidal nti-inflammatory drugs (NSAIDs) and selective inhibtors of prostaglandin H2 synthase (PGHS) 2 because it acks significant antiinflammatory activity. Moreover, s opposed to aspirin, acetaminophen is a poor inhibitor f platelet function at doses that are antipyretic. PGs are generated by the oxygenation of arachidonic cid to the unstable intermediate prostaglandin H2 PGH2) by PGHS, of which there are 2 major isoorms—the constitutive PGHS-1 and the (generally) nducible PGHS-2 (discussed later). These enzymes re also commonly referred to as cyclooxygenase COX) 1 and 2, respectively, in reference to the specific nzymatic active site that catalyzes arachidonic acid xygenation and provides the target for the majority of

Inherited human cPLA2α deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction

Cytosolic phospholipase A2alpha (cPLA2alpha) hydrolyzes arachidonic acid from cellular membrane phospholipids, thereby providing enzymatic substrates for the synthesis of eicosanoids, such as prostaglandins and leukotrienes. Considerable understanding of cPLA2alpha function has been derived from investigations of the enzyme and from cPLA2alpha-null mice, but knowledge of discrete roles for this enzyme in humans is limited. We investigated a patient hypothesized to have an inherited prostanoid biosynthesis deficiency due to his multiple, complicated small intestinal ulcers despite no use of cyclooxygenase inhibitors. Levels of thromboxane B2 and 12-hydroxyeicosatetraenoic acid produced by platelets and leukotriene B4 released from calcium ionophore-activated blood were markedly reduced, indicating defective enzymatic release of the arachidonic acid substrate for the corresponding cyclooxygenase and lipoxygenases. Platelet aggregation and degranulation induced by adenosine diphosphate or collagen were diminished but were normal in response to arachidonic acid. Two heterozygous single base pair mutations and a known SNP were found in the coding regions of the patients cPLA2alpha genes (p.[Ser111Pro]+[Arg485His; Lys651Arg]). The total PLA2 activity in sonicated platelets was diminished, and the urinary metabolites of prostacyclin, prostaglandin E2, prostaglandin D2, and thromboxane A2 were also reduced. These findings characterize what we believe is a novel inherited deficiency of cPLA2.

Effects of reactive gamma-ketoaldehydes formed by the isoprostane pathway (isoketals) and cyclooxygenase pathway (levuglandins) on proteasome function.

Oxidative stress can impair proteasome function, both of which are features of neurodegenerative diseases. Inhibition of proteasome function leads to protein accumulation and cell death. We discovered recently the formation of highly reactive γ‐ketoaldehydes, isoketals (IsoKs), and neuroketals (NeuroKs) as products of the isoprostane and neuroprostane pathways of free radical‐induced lipid peroxidation that are analogous to cyclooxygenase‐derived levuglandins (LGs). Because aldehydes that are much less reactive than IsoKs have been shown to inhibit proteasome function, we explored the ability of the proteasome to degrade IsoK‐adducted proteins/peptides and the effect of IsoK and IsoK‐adducted proteins/peptides on proteasome function. Adduction of IsoK to model proteasome substrates significantly reduced their rate of degradation by the 20S proteasome. The ability of IsoK to inhibit proteasome function directly was observed only at very high concentrations. However, at much lower concentrations, an IsoK‐adducted protein (ovalbumin) and peptide (Aβ1–40) significantly inhibited chymotrypsin‐like activity of the 20S proteasome. Moreover, incubation of IsoK with P19 neuroglial cultures dose‐dependently inhibited proteasome activity (IC50 = 330 nM) and induced cell death (LC50 = 670 nM). These findings suggest that IsoKs/NeuroKs/LGs can inhibit proteasome activity and, if overproduced, may have relevance to the pathogenesis of neurodegenerative diseases.

Acetaminophen inhibits hemoprotein-catalyzed lipid peroxidation and attenuates rhabdomyolysis-induced renal failure

Hemoproteins, hemoglobin and myoglobin, once released from cells can cause severe oxidative damage as a consequence of heme redox cycling between ferric and ferryl states that generates radical species that induce lipid peroxidation. We demonstrate in vitro that acetaminophen inhibits hemoprotein-induced lipid peroxidation by reducing ferryl heme to its ferric state and quenching globin radicals. Severe muscle injury (rhabdomyolysis) is accompanied by the release of myoglobin that becomes deposited in the kidney, causing renal injury. We previously showed in a rat model of rhabdomyolysis that redox cycling between ferric and ferryl myoglobin yields radical species that cause severe oxidative damage to the kidney. In this model, acetaminophen at therapeutic plasma concentrations significantly decreased oxidant injury in the kidney, improved renal function, and reduced renal damage. These findings also provide a hypothesis for potential therapeutic applications for acetaminophen in diseases involving hemoprotein-mediated oxidative injury.

Purification and catalytic activities of the two domains of the allene oxide synthase-lipoxygenase fusion protein of the coral Plexaura homomalla.

The conversion of fatty acid hydroperoxides to allene epoxides is catalyzed by a cytochrome P450 in plants and, in coral, by a 43-kDa catalase-related hemoprotein fused to the lipoxygenase that synthesizes the 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE) substrate. We have expressed the separate lipoxygenase and allene oxide synthase (AOS) domains of the coral protein in Escherichia coli (BL21 cells) and purified the proteins; this system gives high expression (1.5 and 0.3 μmol/liter, respectively) of catalytically active enzymes. Both domains show fast reaction kinetics. Catalytic activity of the lipoxygenase domain is stimulated 5-fold by high concentrations of monovalent cations (500 mmNa+, Li+, or K+), and an additional 5-fold by 10 mm Ca2+. The resulting rates of reaction are ≈300 turnovers/s, 1–2 orders of magnitude faster than mammalian lipoxygenases. This makes the coral lipoxygenase well suited for partnership with the AOS domain, which shows maximum rates of ≈1400 turnovers/s in the conversion of 8R-HPETE to the allene oxide. Some unusual catalytic activities of the two domains are described. The lipoxygenase domain converts 20.3ω6 partly to the bis-allylic hydroperoxide (10-hydroperoxyeicosa-8,11,14-trienoic acid). Metabolism of the preferred substrate of the AOS domain, 8R-HPETE, is inhibited by the enantiomer 8S-HPETE. Although the AOS domain has homology to catalase in primary structure, it is completely lacking in catalatic action on H2O2; catalase itself, as expected from its preference for small hydroperoxides, is ineffective in allene oxide synthesis from 8R-HPETE.

Human colorectal cancer cells efficiently conjugate the cyclopentenone prostaglandin, prostaglandin J2, to glutathione

Cyclopentenone prostaglandins (PGs), particularly those of the J-series, affect proliferation and differentiation in a number of cell lines. J-ring PGs have been shown to be ligands for the peroxisome proliferator-activated receptor (PPAR)-gamma and to modulate NF-kappaB-mediated gene transcription. We have previously reported that large quantities of eicosanoids, including PGJ(2), are produced by the human colorectal cancer cell line HCA-7 while lesser amounts of Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) are formed. In this and other cell lines, cyclopentenone PGs have been shown to increase cell proliferation, but factors that influence their formation and metabolism are poorly understood. Unlike other PGs, cyclopentenone PGs contain alpha,beta-unsaturated carbonyl groups that readily adduct various biomolecules such as glutathione (GSH) in vitro. We now report that in HCA-7 cells, PGJ(2) is largely metabolized by conjugation to GSH. Characterization of the adducts by liquid chromatography (LC)-mass spectrometry (MS) revealed two major metabolites consisting of (1) a novel GSH conjugate in which the carbonyl at C-11 of PGJ(2) is reduced and (2) intact PGJ(2) conjugated to GSH. Approximately 70% of the PGJ(2) added to HCA-7 cells was esterifed to GSH after 2 h of incubation, suggesting this pathway represents the major route of metabolic disposition of PGJ(2) in HCA-7 cells.

Mechanism-Based Therapeutic Approaches to Rhabdomyolysis-Induced Renal Failure

Rhabdomyolysis-induced renal failure represents up to 15% of all cases of acute renal failure. Many studies over the past 4 decades have demonstrated that accumulation of myoglobin in the kidney is central in the mechanism leading to kidney injury. However, some discussion exists regarding the mechanism mediating this oxidant injury. Although the free-iron-catalyzed Fenton reaction has been proposed to explain the tissue injury, more recent evidence strongly suggests that the main cause of oxidant injury is myoglobin redox cycling and generation of oxidized lipids. These molecules can propagate tissue injury and cause renal vasoconstriction, two of the three main conditions associated with acute renal failure. This review presents the evidence supporting the two mechanisms of oxidative injury, describes the central role of myoglobin redox cycling in the pathology of renal failure associated with rhabdomyolysis, and discusses the value of therapeutic interventions aiming at inhibiting myoglobin redox cycling for the treatment of rhabdomyolysis-induced renal failure.

Prostaglandin H2 (PGH2) accelerates formation of amyloid β1-42 oligomers

Epidemiologic evidence implicates cyclooxygenase activity in the pathogenesis of Alzheimers disease, in which amyloid plaques have been found to contain increased levels of dimers and higher multimers of the amyloid β peptide. The product of the oxygenation of arachidonic acid by the cyclooxygenases, prostaglandin H2 (PGH2), rearranges non‐enzymatically to several prostaglandins, including the highly reactive γ‐keto aldehydes, levuglandins E2 and D2. We demonstrate that PGH2 markedly accelerates the formation of dimers and higher oligomers of amyloid β1−42. This is associated with the formation of levuglandin adducts of the peptide. These findings provide the molecular basis for a hypothesis linking cyclooxygenase activity to the formation of oligomers of amyloid β.

Acetylation of prostaglandin H2 synthases by aspirin is inhibited by redox cycling of the peroxidase.

Aspirin exerts its unique pharmacological effects by irreversibly acetylating a serine residue in the cyclooxygenase site of prostaglandin-H2-synthases (PGHSs). Despite the irreversibility of the inhibition, the potency of aspirin varies remarkably between cell types, suggesting that molecular determinants could contribute to cellular selectivity. Using purified enzymes, we found no evidence that aspirin is selective for either of the two PGHS isoforms, and we showed that hydroperoxide substrates of the PGHS peroxidase inhibited the rate of acetylation of PGHS-1 by 68%. Using PGHS-1 reconstituted with cobalt protoporphyrin, a heme devoid of peroxidase activity, we demonstrated that reversal by hydroperoxides of the aspirin-mediated acetylation depends upon the catalytic activity of the PGHS peroxidase. We demonstrated that inhibition of PGHS-2 by aspirin in cells in culture is reversed by 12-hydroperoxyeicosatetraenoic acid dose-dependently (ED50=0.58+/-0.15 microM) and that in cells with high levels of hydroperoxy-fatty acids (RAW264.7) the efficacy of aspirin is markedly decreased as compared to cells with low levels of hydroperoxides (A549; IC50s=256+/-22 microM and 11.0+/-0.9 microM, respectively). Together, these findings indicate that acetylation of the PGHSs by aspirin is regulated by the catalytic activity of the peroxidase, which yields a higher oxidative state of the enzyme.